Podium Presentations for Emerging Technologies

Multi-Site Development of a Real-Time Breast Cancer Recognition and Tumor Metabolic Phenotyping Platform Using Rapid Evaporative Ionization Mass Spectrometry Julia Balog (Presenter) Waters Research Center

To be presented in Track 1 (Steinbeck 1) on Wednesday at 15:30

Introduction.
Oncological surgery is aimed at the complete removal of all cancer cells within a margin of non-cancerous tissue, while minimizing destruction of non-cancerous tissue. Rapid evaporative ionization mass spectrometry (REIMSTM) has been shown to be capable of both detecting tumor cells in real-time, in addition to providing a snapshot of lipid metabolism. If real-time identification of genomic drivers – such as PIK3CA mutation – was possible during surgery, then REIMS could inform clinical decision-making during the course of surgery to avoid positive margins, but also to help inform the selection of adjuvant therapy.

Goals.
Our goal was to (1) harmonize REIMS methods across 3 sites and evaluate cross-site performance of breast cancer recognition models to study the feasibility of real-time margin detection and (2) to investigate the correlation between fatty acid profiles detected in REIMS spectra in normal, wild-type and PIK3CA mutant breast cancer tissues.

Methods.
Human breast tissue was collected at 3 sites (Imperial College London, UK; Maastricht M4I Institute, NL; and Queens University Kingston, CA) affiliated with clinical centers, from patients who underwent surgery for treatment of invasive breast cancer. After macroscopic examination, a local breast pathologist (four pathologists in total) selected tumor and/or normal tissue samples from 6-8 breast cancer surgery cases at each site. A strict protocol for storage and ex vivo REIMS sampling was developed and used for controlled aerosol generation. The evaporated tissue was analyzed with Waters XevoTM G2-XS Mass Spectrometers equipped with REIMS sources. Spectra were acquired in negative ion mode in the m/z 100-1500 mass range. For lipid identification, accurate mass measurements and MS/MS fragmentation were used. Multivariate analysis and subsequent classification were based on principal component analysis combined with linear discriminant analysis (PCA/LDA), while feature selection was performed using linear support vector classifier (lscv). Univariate analysis was done by first performing an F-test for testing the variance of the dataset, followed by a t-test.

Results.
A total of 21 patient samples were collected for the study including 16 invasive ductal carcinoma, 4 invasive lobular carcinoma, 1 ductal carcinoma in-situ (DCIS) and adjacent normal tissue respectively. After histological examination, all sampling points containing solely normal breast tissue or greater than 30% tumor tissue were used to build our database (n=71 cancer and n=139 normal tissue) and was tested at each site separately. Cross validation resulted in 96.4%, 97.11% and 98.57% when building the classifier from single-site or two-site data or using leave-one-patient out cross validation, respectively. The same results could be achieved using only 11 unique species selected by lsvc feature selection. The species selected by the algorithm also contained 3 fatty acids including oleic acid (18:1) enriched in normal tissue and arachidonic acid (20:4) and linoleic acid (18:2) enriched in tumor tissue. Since linoleic acid and arachidonic acid are main components of the omega-6-fatty acid metabolism pathway, we have studied the histological distribution of further 9 relevant fatty acids to further evaluate the upregulation of the omega-6 pathway versus the omega-3 pathway in different cancers. Our findings suggest that while the amount of linoleic acid (18:2) is significantly higher in normal tissue, all other components of the omega-6 fatty acid pathway are significantly (p<0.001) enriched in cancerous tissue including arachidonic acid, dihomo-γ-linoleic acid (DGLA) and FA(22:5). Comparing tumors with PIK3CA mutation to wild type (WT) ones, we also found a significant upregulation of the FA(18:2) - FA(20:2) - FA(20:3) synthetic pathway (p<0.001) leading to a further enhancement of the omega-6 production. In agreement with previous studies, there is also a significant boost in normal tissue towards doxosahexaenoic acid (DHA) showing a better balance of the omega-3 vs omega-6 metabolism when no cancer is present. These observations correlate with previously reported findings showing a significant increase in arachidonic acid levels in tumors linked with an upregulated omega-6 fatty acid metabolism.

Conclusions.
Invasive breast cancer was successfully differentiated from normal breast tissue using real-time mass spectrometric profiling across multiple sites. Recognition models created at each clinical site accurately detected breast cancer at the two other sites, demonstrating the robustness of this approach and algorithms for future clinical use. Furthermore, relative fatty acid concentrations discerned from REIMS were associated with clinically-relevant tumor features such as PIK3CA mutation based on the upregulated omega-6 fatty acid pathway, which can lead to less invasive interventions. Our study of fatty acid metabolism in human tissues correlates to previous work that emphasizes the importance of a balanced omega-3 vs omega-6 diet for the suppression of cancer progression.

Novel aspects.
Cross-site validation of ex vivo breast cancer recognition by REIMS demonstrating applicability to the clinic, fatty acid metabolism monitoring with REIMS for identification of oncogenic PIK3CA mutations.

Direct Swab Analysis by Desorption Electrospray Ionisation – Mass Spectrometry for Preterm Birth Risk Stratification in Patients with Cervical Shortening Katia Capuccini (Presenter) Imperial College London

To be presented in Track 1 (Steinbeck 1) on Wednesday at 15:50

INTRODUCTION
We recently developed Direct Swab analysis by desorption electrospray ionisation – mass spectrometry (DESI-MS). This method permits rapid and direct metabolic profiling of mucosal swab samples whereby spectra can be acquired in less than 2 minutes and without any sample preparation, making it suitable for point-of-care applications. The utility of this method was recently demonstrated in a study of almost 1000 vaginal swabs collected from women during pregnancy which showed that swab profiling by DESI-MS enables rapid and robust prediction of microbiota composition and inflammatory status. This has important clinical implications as the vaginal microbiome and host inflammatory response are modulators of preterm birth risk, which remains the primary cause of death in children under 5 years of age, worldwide. However, the strongest risk factor for preterm birth is a previous preterm delivery. Ultrasound measurement of cervical length in early pregnancy is used clinically as a predictive tool for preterm birth risk. Women with a cervical length ≤ 25mm are considered to be at increased risk of preterm birth and can be treated with a cervical cerclage, which is a procedure whereby a stitch is inserted into the cervix to provide mechanical and biochemical support. This procedure is effective in only subset of women and in some cases, can lead to disturbance of the vaginal microbiome. We hypothesized that metabolic profiling of the cervicovaginal mucosal interface permits assessment of preterm birth risk phenotypes associated with cervical shortening and may facilitate prediction of cervical cerclage effectiveness.

METHODS
Vaginal swabs (BBL CultureSwab MaxV Liquid Amies swabs, Becton, Dickinson and Company, Oxford, UK) were collected longitudinally throughout pregnancy from two study cohorts (VMET, n=160 pregnancies; 437 swabs; VMET II, n=201 pregnancies; 581 swabs). Upon collection, swabs were placed on either Amies transport media or a sterile microcentrifuge tube for transfer and stored at -80 °C until analysis. DESI-MS analysis was performed on an LTQ-Orbitrap Discovery mass spectrometer (Thermo Scientific, Bremen, Germany) with a custom rotating swab holder and source interface designed for direct swab analysis. The DESI-MS sprayer solvent used was a methanol/water (95:5, v/v, HPLC grade Sigma-Aldrich, St. Louis, MO) mixture, with a flow rate of 10 μL/min, a nebulising gas pressure of 7 bar, and a sprayer voltage of 4.3 kV. For each swab, approximately 30 spectra were acquired in positive and negative ionisation model with a m/z window from 50–1000. MS spectra were pre-processed (peak picking and grouping) in R with the maldiquant package. Multivariate partial least squares-discriminant analysis and random forest models with K-fold cross-validation were used to assess the ability of DESI-MS metabolic profiles to predict clinical outcomes.

RESULTS
Multivariate analysis performed on both VMET and VMET II samples indicated that DESI-MS cervicovaginal metabolic profiles are weakly predictive of previous pregnancy history (previous PTB n=204, vs previous cervical treatment n=109; AUC=0.65 negative mode, AUC=0.64 positive mode). Similarly, DESI-MS profiles provided a weak prediction of subsequent treatment for cervical shortening (n=85) compared to controls matched for gestational age and microbial composition (n=85) (positive ion mode AUC=0.65, negative ion mode AUC=0.67). DESI-MS profiling of samples collected following intervention indicated capacity to distinguish women who went on to subsequently shorten their cervix despite cerclage (n=14) from those who maintained cervical length (n=37) (AUC =0.74 negative mode, AUC=0.62 positive mode). Finally, the capacity of DESI-MS to predict vaginal microbial composition in women with a cervical cerclage in situ was found to remain strong at genera level (AUC=0.92 negative mode, AUC=0.93 positive mode) and for major species-level community state types (Lactobacillus crispatus dominated v Lactobacillus iners dominated, AUC= 0.81 negative mode, AUC=0.84 positive mode; Lactobacillus crispatus dominated v dysbiotic, AUC=0.98 negative mode, AUC=0.98 positive mode and Lactobacillus iners dominated v dybiotic, AUC=0.93 negative mode, AUC=0.94 positive mode).

DISCUSSION
Our data indicate that early pregnancy cervicovaginal fluid metabolic profiles obtained using Direct Swab Analysis by DESI-MS are not strongly reflective of background preterm birth history risk phenotypes. However, they offer potential for stratifying women who will subsequently experience cervical shortening and require clinical intervention. This indicates that early biochemical changes associated with pathophysiological shortening of the cervix are detectable in the cervicovaginal metabolome prior to clinically overt shortening detectable by ultrasound. Early detection of women at risk of cervical shortening may improve efficacy of current strategies for its treatment and prevention. Moreover, our results suggest that swab profiling by DESI-MS may also permit monitoring of patient response and prognosis following cervical cerclage. Insertion of cervical cerclage does not hamper the ability of the method to robustly predict vaginal microbiome composition, which is an important mediator of maternal and neonatal health outcomes during pregnancy. Collectively, these findings highlight Direct Swab analysis by DESI-MS as an innovative approach for the stratification of preterm birth risk phenotypes associated with cervical shortening and as a potential tool for the monitoring and prediction of response to commonly used preventative interventions.

Rapid and Sensitive Protein Quantitation in Biofluids by Paper Spray Mass Spectrometry: Single Instrument Albumin/Creatinine Ratio Measurements Chris Gill (Presenter) Vancouver Island University

To be presented in Track 1 (Steinbeck 1) on Wednesday at 16:10

INTRODUCTION: Direct mass spectrometry approaches such as paper spray mass spectrometry (PS-MS) are presenting new alternatives as candidate methods for clinical workflows. PS-MS, in particular, offers a facile strategy for chemical measurements in complex samples such as biofluids. Small aliquots (i.e., ≤10µL) of sample are deposited on pointed paper strips with co-deposited internal standards. The strips are moistened with a suitable solvent, and upon the application of high voltage, ions are generated in a manner akin to electrospray, allowing direct analyte quantitation via tandem mass spectrometry. The strips are inexpensive and disposed for each measurement, eliminating carryover, and can be used to conduct ‘on-paper’ derivatization reactions as well as replace the extraction/preconcentration steps necessary in other analytical workflows.

OBJECTIVES: To demonstrate the use and effectiveness of chemically modified PS-MS paper substrates for the rapid, sensitive, and quantitative measurement of proteins in biofluids, including the rapid ‘one instrument’ measurement of albumin/creatinine ratios in urine.

METHODS: All measurements were performed by paper spray tandem mass spectrometry with a high-throughput paper spray ion source (Thermo Scientific™ TSQ Altis™ triple quadrupole mass spectrometer with a VeriSpray™ source). PS-MS paper substrates were modified by grafting polyamidoamine (PAMAM) dendrimers to their surface, increasing protein affinity. The extraction / preconcentration of target proteins from urine and serum was accomplished by vortexing the strips in 150 µL biofluid samples, followed by a short wash. Measurement by PS-MS was accomplished using barcode traceable VeriSpray™ PS-MS sample plates, allowing multiplexed measurements of 24 strips per plate, and the unattended measurement of up to 240 samples. Quantitative calibrations were achieved for both proteins and creatinine utilizing co-deposited internal standards.

RESULTS: The PAMAM functionalized PS-MS paper substrates were characterized for direct protein measurements in human urine and serum sample matrices. This included optimizing sample extraction times, washing protocols and evaluating the storage stability of the modified paper substrate. A 30 second protein vortex extraction followed by a 5 second acetonitrile wash was optimal. Once prepared, PAMAM functionalized PS-MS substrate exhibited no deterioration in performance when stored at ambient conditions over a one month period. As examples, the direct measurement of hemoglobin and albumin in urine and lysozyme and insulin in serum is presented, achieving up to 11 fold sensitivity improvements, dependent upon the charge state of the protein. The analytical performance of the functionalized paper substrate was evaluated for the analysis of albumin in urine, achieving linearity with R2 > 0.99, LOD of 1.1 µg mL-1, LOQ of 3.8 µg mL-1, precision <10%, and recoveries of 70-83%. The direct, simultaneous measurement of urinary albumin and creatinine ratios for anonymous patient samples by PS-MS demonstrates excellent comparison with validated clinical results obtained utilizing immunoturbimetry for albumin and the spectrophotometric Jaffe method for creatinine.

CONCLUSION: The use of PAMAM functionalized PS-MS paper substrate enhances the sensitivity of direct protein quantitation from biofluids. The ‘one instrument’ quantitative measurement of urinary albumin/creatinine ratios by PS-MS shows significant promise as a new candidate method for clinical analyses.

Redefining Serological Diagnostics with Immunoaffinity Proteomics Andrei Drabovich (Presenter) University of Alberta

To be presented in Track 3 (Steinbeck 3) on Wednesday at 15:30

INTRODUCTION: Serological diagnostics relies on identification of disease-specific antibodies in serum and is an essential tool in clinical diagnostics. Current serological tests utilize immunoassays and focus on fast and convenient assay development and high throughput measurements. Limitations of such tests include semi-quantitative measurements, lack of standardization, cross-reactivity, and inability to distinguish between human immunoglobulin subclasses. Advances in affinity proteomics and standardization of protocols, including our recent studies [1-4], facilitated development of sensitive and reproducible assays for quantification of low-abundance proteins and antibodies.

OBJECTIVES: We suggested that immunoaffinity proteomics may advance serological diagnostics of infectious diseases and cancer through the rational design and standardization of assays, and identification and quantification of disease-specific clonotypes of human antibodies, thus paving the way for precision immunology.

METHODS: Our approach for the rational design of serological assays utilizes immunoprecipitation of the antigen-binding human antibodies from blood serum, plasma or saliva followed by differential quantification of human antibody isotypes (IgG, IgA, IgM, IgE, IgD) and subclasses (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) with targeted mass spectrometry. Trypsin-cleavable and heavy isotope-labeled synthetic peptide internal standards targeting the Constant Heavy chains of human antibodies enable their absolute quantification (ng/mL). Simple assay design, targeted mass spectrometry and fast microflow separations provide high reproducibility, sensitivity and throughput (120 samples/day). Concurrent immunoaffinity-shotgun proteomics provides identification of immunoglobulin Fc interactomes and enables identification and quantification of antigen-specific immunoglobulin clonotypes. Our assays were validated in COVID-19 and prostate cancer patient samples.

RESULTS: A multiplex immunoprecipitation-selected reaction monitoring (IP-SRM) assay enabled differential quantification of anti-SARS-CoV-2 antibody isotypes and subclasses in blood serum, plasma and saliva. Likewise, an immunoprecipitation - parallel reaction monitoring (IP-PRM) assays quantified NCAP_SARS2 protein with a limit of detection of 313 pg/mL in serum. Evaluation of 36 antigen-antibody subclass combinations revealed receptor-binding domain (RBD)-IgG1 as a combination with the highest diagnostic specificity and sensitivity. Further validation revealed that anti-RBD IgG1, IgG3, IgM and IgA1 levels were significantly elevated in COVID-19- convalescent plasma and saliva, while IgG2, IgG4, IgA2 levels were not informative [1]. Anti-RBD IgG1 revealed a diagnostic cut-off of 408 ng/mL and provided 99.3% diagnostic specificity at 88% sensitivity to detect COVID-19 convalescent plasma. Evaluation of IgG1+IgA1+IgM combination in negative (N=143) and positive convalescent (N=82) plasma revealed 100% diagnostic specificity at 96.3% sensitivity. Interestingly, immunoaffinity-shotgun proteomics identified co-precipitation of immunoglobulin interactome (C1q complement complexes) and revealed IGHV3-7/IGKV3-20 as one of the most abundant clonotypes of anti-RBD antibodies circulating in plasma of COVID-19 patients. Finally, immunoaffinity proteomics platform enabled evaluation of serological response to prostate-specific proteins circulating in serum of patients with prostate cancer.

CONCLUSIONS: Immunoaffinity proteomics as a platform for serological diagnostics will facilitate standardization and improvement of the existing serological tests, enable rational design of novel tests, and offer novel tools for precision immunology, investigation of antibody subclass cooperation in immunity response and de novo sequencing of circulating high-affinity antibodies.

REFERENCES:
[1] Fu, Z.; Rais, Y.; Dara, D.; Jackson, D; Drabovich, A.P. Rational Design and Development of SARS-CoV-2 Serological Diagnostics by Immunoprecipitation-Targeted Proteomics. Analytical Chemistry 2022, 94, 12990–12999;
[2] Fu, Z.; Rais, Y.; Bismar, T.A; Hyndman, ME; Le, XC; Drabovich, AP. Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein by Orthogonal Immunoprecipitation-Mass Spectrometry Assays. Molecular & Cellular Proteomics, 2021, 20, 100075;
[3] Drabovich, A.P.; Saraon, P.; Drabovich, M.; Karakosta, T.D.; Dimitromanolakis, A.; Hyndman, E.; Jarvi, K.; Diamandis, E.P. Multi-omics Biomarker Pipeline Reveals Elevated Levels of Protein-glutamine Gamma-glutamyltransferase 4 in Seminal Plasma of Prostate Cancer Patients. Molecular & Cellular Proteomics, 2019, 18, 1807;
[4] Schiza, C.; Korbakis, D.; Jarvi, K.; Diamandis, E.P.; Drabovich, A.P. Identification of TEX101-associated Proteins Through Proteomic Measurement of Human Spermatozoa Homozygous for the Missense Variant rs35033974. Molecular & Cellular Proteomics, 2019, 18, 338.

Intra-operative Margin Assessment by REIMS During Breast Cancer Surgery and Validation Using a Spatio-Temporal Navigated Cautery and Histopathology Martin Kaufmann (Presenter) Queen’s University

To be presented in Track 1 (Steinbeck 1) on Thursday at 8:45

Introduction
Rapid evaporative ionization mass spectrometry (REIMS) can profile and classify tissues without sample preparation in real-time, using pre-built spectral databases and machine-learning. REIMS has been proposed as an intraoperative tool to inform surgeons during breast tumor removal, to reduce the need for re-operation due to remaining cancer cells on the periphery (positive margins), which occurs in approximately 25% of cases. While REIMS in its current form can indicate the presence of cancerous tissue, it does not indicate 3D location. This limits the ability to validate mass spectra obtained intraoperatively, and in the future, its ability to inform decision-making in the operating theatre.

Objectives
In the current study, we combined REIMS with a spatio-temporal navigated cautery that was temporally-synched to REIMS. We tested this platform intraoperatively on 22 breast cancer (BCa) surgery cases, and retrospectively compared the margin status determined by the navigated REIMS system with that of the ‘gold standard’ histopathology report.

Methods
A multivariate model based on PCA/LDA was trained and cross-validated on REIMS spectra sampled from 11 pathology-validated ex vivo BCa surgery specimens, including N=118 spectra from normal breast adipose, and N=36 spectra from invasive BCa. During 22 surgeries, REIMS was used to acquire testing data from tissue dissected with an electromagnetically-tracked cautery, and surgeons would call-out tissue types being dissected. Spectra were retrospectively classified using the above model. Prior to surgery, a localization wire with an electromagnetic sensor was placed into the tumor as a marker and a 3D map of the tumor was contoured using tracked ultrasound. REIMS was temporally-synched with the 3D tracker, allowing computation of the spatial origin of spectra using the time of acquisition. Spectra classified as BCa were mapped onto a display of the tumor region and compared with the pathology report.

Results
Spectra from ex vivo BCa specimens were characterized by a distinctly elevated ratio of glycerophospholipids (PL)-to-triglycerides (TG) as compared with normal breast adipose. A PCA/LDA-based classifier exhibited >90% accuracy on cross-validation. In the intraoperative patient cohort, 4/22 cases had positive margins as assessed by pathology, all of which were correctly identified by REIMS. Notably, two of these cases were positive for ductal carcinoma in situ with the location of the positive margin matching the pathology report. Intra-operative spectra classified as BCa exhibited similar PL:TG ratios to that observed in the ex vivo data. Further, 18/22 cases were margin negative, and observations from 12 of those cases were consistent with the histopathology assessment. There were 6 margin-negative cases that were determined to have positive margins by REIMS (‘false positive’). As well, 4/6 false positive cases were noted by histopathology as ‘close margins’ with cancer cells being detected within 1 mm of the inked margin or noted by the surgeon as containing dense but otherwise normal breast tissue. Other normal tissue types dissected during breast surgery with high PL content were misclassified as BCa, including skin and muscle, but these spectra can be rationalized by relative distance from the tumor region using the navigation data and/or call-outs from the surgeon.

Conclusions
Our study points to the importance of spatio-temporal tracking to help validate intraoperative tissue characterization by REIMS profiling during BCa surgery. We anticipate that a navigated REIMS platform will eventually help inform the surgeon of specific locations where a wider excision may be necessary to avoid a positive margin.

Integrated Morphometric and Molecular Classification of Central Nervous System Cancers Using a Unified Platform with Picosecond Infrared Laser Mass Spectrometry Alexa Fiorante (Presenter) University of Toronto

To be presented in Track 1 (Steinbeck 1) on Thursday at 9:05

Introduction
Based on our growing understanding of the molecular heterogeneities in the central nervous system cancers, the recent ‘World Health Organization Handbook for Diagnosis of Central Nervous System (CNS) Cancers’ proposes an ‘integrated’ molecular approach to supplement morphometric indicators traditionally used in rapid intraoperative CNS diagnosis methods. The bulk of the proposed molecular diagnosis methods, however, are based on genomic or immunohistochemistry assays and as such cannot be used on intrasurgical timescales to drive personalized cancer resections in the operating theatre. Therefore, there is a need for a unified methodological platform that can deliver rapid CNS cancer diagnosis based on both morphometric and molecular indices of interest on intrasurgical timescales. This is crucial for CNS cancers where preoperative biopsies are uncommon and there is a growing body of retrospective data suggesting benefit to maximal resection in patients with cancers of certain molecular types, currently only actionable in revision surgeries.
Capitalizing on the close coupling between lipid metabolism and cancer formation & progression, untargeted mass spectrometry profiling of the tissue lipidome using ambient ionization methods has constituted an attractive strategy for rapid determination of cancer types through comparing rapidly acquired MS1 profiles of a query specimen to a validated library of known cancer lipid fingerprints. One such method is Picosecond InfraRed Laser Mass Spectrometry (PIRL-MS) that has been successful in discriminating molecular subgroups of medulloblastoma cancers in 10 seconds with minimal tissue consumption (< 1mm3) and no thermal damage outside the sampling zone.

Methods
A local tissue bank containing over 3,000 frozen specimens of over 20 different classes of adult and pediatric brain cancers has been subjected to analysis by PIRL-MS towards building a molecular library of PIRL-MS signatures of morphometrically or molecularly distinct classes of CNS cancers. 10-second MS1 PIRL-MS spectra are collected on a Waters Xevo G2 XS quadrupole Time of Flight (qTOF) mass spectrometer. Multivariate modeling of the MS1 spectra using both supervised dimensionality reduction methods as well as unsupervised clustering are conducted to validate the molecular models using blind sample predictions for the determination of sensitivity and specificity of use, and to inform of potential lead mass-to-charge (m/z) values most important for cancer class differentiations. These lead values are subjected to rigorous determination of their molecular identities using targeted high-resolution tandem mass spectrometry after chromatography on a Synapt qTOF (Waters). The resultant list of the characterized tissue metabolites will be tested again with a ‘sparse’ multivariate clustering approach for the determination of the list’s success rate in 10-second classification of morphometric and molecular classes of common CNS cancers. The use of a well-characterized metabolite array for MS1 cancer type classification is expected to shield against noise that has been reported in many untargeted MS explorations of cancer molecular fingerprint and in related metabolomics endeavours as a confounding factor.

Results
We have investigated 150 pediatric brain cancers (3 morphometrically distinct types and 7 molecularly distinct types) and close to 500 adult brain cancers (14 morphometrically distinct classes and so far only 2 molecularly distinct type of one class). The sensitivity and specificity of the integrated morphometric and molecular diagnosis for pediatric brain cancers with 10-second PIRL-MS was > 94%. This classification used 18 tissue lipids whose identities were determined with targeted chromatography and tandem mass spectrometry. The classification of the adult brain cancers with PIRL-MS is currently ongoing but preliminary data suggests > 84% sensitivity and specificity across 14 morphometric classes and select molecular types of isocitrate dehydrogenase-1 or BRAF-V600E mutations with only 10 seconds of data analysis and multivariate model comparisons. Further refinement of the model and blind sample tests will take place in the near future. We will also run post PIRL MS sampling immunohistochemistry to confirm the tumour class. We suspect our numbers will improve after this is done. Some of the 16% that were not classified correctly may not be the correct tumour in the frozen sample.

Conclusions
The integrated diagnosis workflow proposed by WHO heavily emphasizes the use of multiple instruments with varying turnaround times. The demonstrated capability of PIRL-MS in delivering 10-second diagnosis of both morphometrically distinct as well as molecularly different (sub)types of CNS cancers contributes to its potential to form a unified platform for integrated morphometric and molecular diagnoses of CNS cancers on the same instrumental platform. This integration of diagnostic workflow that now can use a single instrument for diagnosis based on molecular and morphometric indicators (from their correlated downstream molecular metabolites) is expected to save costs and improve the current standard of care, offering benefits to both patients and hospitals that may adopt it upon authorization for future clinical use.

Molecularly Aware Robotics for Surgery (MARS) – Autonomous Surgical Intervention Using Mass Spectrometry Mapping Mark Runciman (Presenter) Hamlyn Centre for Robotic Surgery, Department of Surgery and Cancer, Imperial College London

To be presented in Track 1 (Steinbeck 1) on Thursday at 9:25

INTRODUCTION:
Achieving negative margins is important in surgical cancer treatment; however, there is a trade-off between achieving a negative margin and preserving healthy tissue. Mass spectrometry -based tools are becoming available that enable classification of in vivo tissue in real time to improve margin definition. In tandem, robotic surgical platforms are being developed that enable precise and repeatable motion control.

The objective of this work was to demonstrate a compact soft robotic platform with autonomous localisation and guidance capabilities based on mass spectrometry classification data.

METHODS:
A hybrid soft robot was used to guide an optical fibre and collection tube, for laser ablation of tissue and subsequent collection of the aerosol produced. A 10600 nm CO2 laser (Omniguide, USA) was used for ablation, while a mass spectrometer (Xevo G2-S QToF, Waters Corporation, USA) coupled with a Rapid Evaporative Ionization Mass Spectrometry (REIMS) source was used for molecular analysis of the aerosol. The mass spectral results are processed into tissue models using multivariate statistical analysis (Principal Component Analysis &ndash; Linear Discriminant Analysis) which models are used for quasi-real (&gt;100 ms) time tissue classification. The hybrid soft robot is a combination of two robotic mechanisms, capable of gross and fine positioning respectively, and three cameras for both monocular tool tracking and stereoscopic tissue surface reconstruction.

The gross positioning system consisted of a soft robotic parallel mechanism with a large workspace, which guides the base of a thermally actuated fibre robot, the fine positioning system. Tracking of the gross positioning robot was achieved using computer vision; specifically, a bespoke marker on the robot shaft was tracked using graph-based deep learning methods that enabled sub-millimetre accuracy. As such, the pose of the robot tip was tracked while mass spectrometry data was collected to produce 3D point clouds.
To autonomously steer the robot, the direction of motion of the soft robotic platform was altered depending on the classification data from the mass spectrometry instrument. Algorithms were designed such that the robot first identified and then followed the boundaries between healthy and unhealthy tissue.
The autonomous guidance and localisation capabilities were demonstrated in a series of experiments on mouse skin tissue containing cancerous regions.

RESULTS:
The hybrid robot platform successfully produced mass spectrometry point clouds of several ex vivo mouse skin tissue samples, each containing multiple cancerous lesions. Based on the classification mappings, the robot was able to identify boundaries of the cancerous regions, generate trajectories in 3D space, then execute these trajectories while ablating the tissue.

The initial tissue models consisted of thin sections (mouse brain, pork liver) mounted on glass slides and were analysed by the robotic platform coupled with a REIMS-equipped mass spectrometer. Based on the molecular profiles (which mainly consist of metabolites, lipids and other small molecules), the structures of the hippocampal region were successfully identified.

One of the main advantages of the robot is its small size and flexibility. The robotic platform can be utilised to analyse normally hard-to-reach areas such as the head and neck area, or internal body cavities such as colorectal areas, where it will have the capability to autonomously perform molecular profile-based surgical interventions. Live animal experiments were performed in pigs to demonstrate the possibility of applying the system in an in vivo environment.

Due to the molecular pathology-based decision-making of the system, the robot has great potential for successful resection of diseased tissues where the preservation of function is important (such as skin cancer on the face, or head and neck area). The setup was successfully used to analyse the head and neck area of a human cadaver to demonstrate the capability to sample deep tissue areas without causing significant disruption to the surrounding tissues.

Currently ex vivo tissue collection and analysis is being prepared for skin cancer and cervical cancer samples. Ethical approval is being sought for the in vivo demonstration of the robotic system, for skin cancer and cervical cancer cases. The robot was able to autonomously ablate cancerous tissue while preserving healthy tissue.

DISCUSSION:
This work demonstrates the feasibility of autonomous surgical interventions using mass spectrometry data to guide a robotic tool. The soft robotic device used has multiple potential applications, including endoluminal minimally invasive interventions, for example, which will be the focus of this project in the future. Furthermore, the robot hardware can be manufactured rapidly and economically, increasing the accessibility of this technology.

Decreasing Relapse in Esogastric Cancer by Improved Diagnostic with Spidermass Technology Léa Ledoux (Presenter) Laboratoire Prism Inserm U1192

To be presented in Track 2 (Steinbeck 2) on Thursday at 9:05

Aim:
With about 951,000 cases each year, esogastric cancer (EC) is the fifth most often-diagnosed cancer worldwide. EC encompasses a variety of different carcinoma subtypes among which the Poorly Cohesive Carcinoma (PCC) is a very aggressive type occurring in young patients. PCC represents a diagnostic challenge because of its diffuse character and therefore, is difficult to distinguish intraoperatively leading to a relapse of a half of the patients. Our objective is to setup an accurate intraoperative diagnostic to assist surgeons in the uptake of PCC using the SpiderMass technology. To this end MALDI-MSI was used to depict the tissue heterogeneity and reveal specific lipid profiles of PCCs by comparison to other ECs using SpiderMass.

Methods:
A cohort of EC excised tissues were sectioned on a cryostat (Leica CM 1510S). Three sections were collected, one (5 µm) for the H&E staining, the second (20 µm) for SpiderMass analysis and the last one (12 µm) for the MALDI-MSI. The MALDI-MSI analysis was performed in both polarities on a MALDI-TOF (Rapiflex, Bruker) at 50 µm spatial resolution using norharmane as matrix (7 mg/mL). The SpiderMass analysis was performed on a Q-TOF instrument (Xevo, Waters) in both polarities. The data were processed using SCILS (MALDI-MSI and SpiderMass) and AMX (SpiderMass). LDA was used to build classification models and the model was assessed by interrogation in blind on a validation cohort of 50 tissues.

Results:
The 136 (68 healthy and 68 cancers) EC sections that were analyzed using MALDI-MSI showed a different and specific molecular profile for each type of tissue, enabling a clear discrimination between them. Because of the strong similarity between the MALDI-MSI and the SpiderMass-MSI data (93% in negative ion mode), the same cohort was analyzed with the SpiderMass. SpiderMass images were very like the MALDI ones leading to the identification of the same discriminative markers. The markers discriminating the different types and subtypes were identified by MS2 and manually annotated. The SpiderMass data were then used to build classification models for typing and subtyping the EC using machine-learning and deep-learning. The models were further validated in blind on an extra cohort of 50 tissues.

Discussion:
Thanks to the training cohort (136 tissues) and the validation cohort (50 tissues), we were able to build a very trustful classification model for discriminating cancer from healthy tissues first, but also to subtype the esogastric cancer. Thanks to this proof-of-concept we will be able to further evaluate the SpiderMass as a fast intraoperative diagnostic and prognostic tool in the pathology lab for direct comparison to the gold standard histology.

Targeted Quantification of Androgen Metabolites Using Ion Mobility-Mass Spectrometry Christopher Chouinard (Presenter) Clemson University

To be presented in Track 1 (Steinbeck 1) on Thursday at 10:00

INTRODUCTION:
Identification and quantification of steroid metabolites can be confounded by significant isomeric heterogeneity, compounded by other analytical challenges associated with steroid analysis (poor ionization efficiency, wide polarity range, etc.). Ion mobility-mass spectrometry (IM-MS) has shown promise as a rapid approach (acquisition times <100 ms) that can easily be coupled with existing chromatographic methods. Herein we will demonstrate a targeted LC-IM-MS/MS method for the quantification of androgen metabolites in human urine.

OBJECTIVES:
The primary objective of this study was to demonstrate simultaneous quantification of nearly forty androgen metabolites in human urine, with a reduced emphasis on chromatographic separation of isomers.

METHODS:
A targeted LC-IM-MS/MS method was developed for targeted quantitation of androgen metabolites in human urine. A simple 2-minute chromatographic gradient was chosen to highlight the rapid separation of isobaric/isomeric species with IM. Urine samples were extracted by SPE and injected onto the LC-IM-MS/MS system, where data was collected in multiplexed acquisition mode. All data was processed using a streamlined workflow software workflow culminating in quantification with Skyline. Confirmation for each metabolite was provided by retention time, collision cross section (CCS), and exact mass. Quantitation was performed over a clinically relevant concentration range for the analytes of interest. All data were acquired using either Agilent 6560 or MOBILion Systems MOBIE HRIM IM-MS platforms.

RESULTS:
Traditional GC- and LC-MS/MS methods for quantification of steroid metabolites rely on chromatographic approaches for separation of isobaric/isomeric species. Herein, we demonstrated that a fast RP-LC method (2 mins), which in many cases did not provide adequate separation, could be easily coupled with IM-MS/MS to provide rapid gas-phase separation by ion mobility. Nearly 40 androgens and their metabolites were first analyzed individually to populate a target library with retention time, CCS, and exact mass for each ion adduct identified. CCS values ranged from 166-190 Å2 for protonated species and 193-207 Å2 for sodiated species; these values were highly reproducible (RSD ≤ 0.5%), allowing improved confidence of identification. Human urine was then analyzed to detect and quantify these metabolites and determine limits of detection and sensitivity over a concentration range from 1 pg/mL to 10 ng/mL; preliminary results (Velosa et al. JASMS, 2022) demonstrated an LOD of ~500 pg/mL for testosterone.

CONCLUSION:
IM-MS/MS was demonstrated as a rapid method for detection and quantification of androgen metabolites in human urine. A fast chromatographic gradient was coupled with the developed IM-MS/MS method, such that isobaric/isomeric resolution was not reliant upon chromatographic differentiation but instead by mobility. Skyline was employed as a streamlined data processing approach for quantification. We hope to expand this workflow to other molecular classes relevant to the clinical lab, including opioids and other drugs of abuse.

PFAS Dark Matter and Slippery Cannabis: Disparate Problems with a Similar Path to a Solution Frederick Strathmann (Presenter) MOBILion Systems

To be presented in Track 1 (Steinbeck 1) on Thursday at 10:20

INTRODUCTION:
PFAS compounds have a long and diverse history of applications yet only recently has sufficient attention been focused on the environmental and toxicological impacts of their use. The number of PFAS compounds has expanded rapidly and it is estimated that 5,000 to 10,000 PFAS compounds exist. Despite the tremendous numbers of PFAS compounds in existence, a relatively small number have been studied in depth and are commercially tested routinely. The term “PFAS Dark Matter” has emerged to signify the recognized gap between Total Organic Fluorine, Total Oxidizable Precursors, and targeted methods using tandem mass spectrometry for PFAS concentration assessment. It is estimated that nearly every individual has at least one PFAS present in their blood, and the toxicological significance of PFAS and the implications of the PFAS Dark Matter are far from being fully appreciated. Similarly multifarious, cannabis has a complex chemical composition that includes terpenes, sugars, hydrocarbons, steroids, flavonoids, amino acids, and other compounds of potential interest. More than 700 natural constituents have been identified and more than 100 are classified as cannabinoids. The toxicological community has been challenged with the appearance of isomers of various cannabinoids causing numerous analytical challenges with limited solutions beyond chromatographic run time extension. Though diverse in the context of the challenges presented, trends in PFAS and cannabinoid production and laboratory-associated detection methods are quickly evolving in a manner reminiscent of Novel Psychoactive Substances with similar complexities involving identification, testing, and interpretation of toxicological data.

OBJECTIVES:
The primary objective of this study was to leverage a relatively novel analytical combination of LC, HRIM and QTOF approaches to unravel the complexity seen with existing separation challenges. A secondary objective of this study was to call attention to the need for clinical laboratories to evolve beyond existing workflows in anticipation of the challenges with emerging analytical needs associated with higher complexity biomarkers on the horizon.

METHODS:
We used the MOBILion HRIM system based on Structures for Lossless Ion Manipulation (SLIM) to assess cannabinoids and PFAS in a variety of matrices. A combination of Flow Injection Analysis or Liquid Chromatography with HRIM was used prior to detection using an Agilent QTOF. Accurate mass, isotope spacing, isotope ratios, and mobility aligned fragmentation were used in various combinations for tentative and absolute identification depending upon available standards. In several cases, CCS values were derived providing a unique, molecular identifier that was leveraged to generate 2 dimensional plots of CCS vs. m/z to elucidate trendlines and characteristic subclasses revealing distinctive relationships within and across compound classes. Lastly, previously established CCS values were used to generate reference plots of CCS vs. m/z as a tool to understand potential impact of interferences with known, endogenous compounds where applicable.

RESULTS:
Herein we report our use of the MOBIE® high-resolution ion mobility system (HRIM) from MOBILion with an Agilent LC-QTOF system to resolve fourteen different cannabinoid species reported in cannabis including positional isomers delta-8 and delta-9 THC, with an approximately 0.4% CCS difference, sufficiently resolved in matrix-free samples in the absence of chromatographic separation. Eight previously identified perfluorooctane sulfonates including the tentative identification of one additional branched form previously unseen, were found with enhancement of existing chromatographic separation by the HRIM system. Lastly, we explored an emerging link between hemp and PFAS in a subset of available extracts as a potential consequence of phytoremediation efforts with implications into as yet unknown toxicological significance.

CONCLUSION:
The LC-HRIM-QTOF system used is a powerful combination that can be implemented to enhance existing LC workflows for deeper sample characterization, reduce LC reliance to boost throughput, and add CCS values to existing compound identification and classification approaches. The examples provided here are relatively simple analytical challenges where existing separation technology has been limited in its utility for long-term, routine use. The clinical laboratory field has been eagerly watching the various “omics” fields with the anticipation of multiomics, diagnostic test availability, yet we have been largely idle in integrating truly novel analytical techniques outside of molecular testing. As the intricacies of each of the omics fields continues to be unraveled, novel technologies being applied to applications residing on the outskirts of existing and established workflows hold promise for meeting the ever-increasing complexity of novel testing expectations to come.

Leveraging Ion Mobility-Mass Spectrometry for High-throughput Multi-omics Kelly Hines (Presenter) University of Georgia

To be presented in Track 1 (Steinbeck 1) on Thursday at 10:40

Introduction
There is growing interest and appreciation in the use of mass spectrometry-based multi-omics approaches to study biological processes and diseases from a systems-level perspective. The performance of discovery-level multi-omics typically involves the partitioning of a complex sample into its individual components, followed by a thorough analysis of each “ome” under optimized LC and MS conditions. However, these multi-omics experiments must be streamlined before their findings can be implemented into diagnostic or prognostic applications. The rapid gas-phase structural separations afforded by IM-MS provide an opportunity for high-throughput measurements of biological samples containing mixtures of lipids, metabolites, peptides, and other biochemicals.

Objectives
We are developing methods for high-throughput multi-omics based on flow injection analysis (FI) and ion mobility-mass spectrometry (IM-MS). The feasibility and advantages of FI-IM-MS will be demonstrated for the identification of microorganisms to the species and strain levels using integrated lipidomic and metabolomic features.

Methods
Pseudomonas aeruginosa (n=3), Acinetobacter baumannii (n=3), and Staphylococcus aureus (n=7) strains were cultured overnight in tryptic soy broth, with six replicates per strain. Bacteria suspensions in sterile saline solution were adjusted to achieve a consistent cell density and an equal volume was used to obtain pelleted sample via centrifugation. One set of replicates (n=3) was extracted using the Bligh & Dyer two-layer liquid-liquid extraction method, from which the aqueous and organic layers were collected. The second set of replicates (n=3) was extracted using simplified, single-phase extraction based on acetonitrile, methanol and water. The three sets of extracts (i.e., lipids from Bligh & Dyer, metabolites from Bligh & Dyer, and lipids+metabolites from single-phase) were analyzed first by hydrophilic interaction liquid chromatography (HILIC) coupled to a traveling wave ion mobility-mass spectrometry (TWIM-MS), and then by flow-injection (FI) analysis couple to TWIM-MS. Data was collected in positive and negative ionization modes. Data were analyzed using Progenesis QI.

Results
In comparing the HILIC and FI-IM-MS methods, we observed a strong positive correlation between peaks areas for the same analytes in both methods. The FI-IM-MS method yielded RSDs below 15% for 95% of the features detected in replicate injections of a pooled mixture. In all extractions and analyses, the lipid and/or metabolite profiles were sufficient to separate the microorganisms based on their Gram stain status (positive or negative) and to the species level in principal components analyses (PCA). Although fewer total features were detected from the FI-IM-MS using the same significance threshold, the same individual metabolites and lipids were identified as major contributors to the group differences in the PCA. The combination of both lipids and metabolites from the single-phase extraction method improved the separation of strain-level differences within all three organisms. Lipids and metabolites that contributed to the PCA separation from single-ome experiments were among the features contributing to the PCA clustering in the single-phase, multi-ome dataset.

Conclusion
Our preliminary evaluation of the FI-IM-MS approach to multi-omics has demonstrated that combining lipid and metabolite experiments into a single method improves the resolution of microorganism identifications to the strain level without sacrificing throughput or precision.

The Power of Mass Spectrometry in the Care of Patients with Monoclonal Gammopathies Mindy Kohlhagen (Presenter) Mayo Clinic

To be presented in Track 1 (Steinbeck 1) on Thursday at 14:40

Objective: To demonstrate the roles of MALDI-TOF and ESI-TOF mass spectrometry in monitoring patients with monoclonal gammopathies.

Introduction: Over the past few years, our lab has transitioned the majority of clinical testing for serum M-proteins from gel-based immunofixation to immunoaffinity purification with MALDI-TOF MS analysis (Mass-Fix). Mass-Fix performs comparably to immunofixation, but with several important advantages. Mass spectrometry allows for improved M-protein tracking, detection of clinically relevant post-translational modifications of M-protein light chains, and distinguishes therapeutic monoclonal antibodies (tmAbs) from endogenous M-proteins with increased analytical sensitivity and specificity. MALDI-TOF MS has rapid high throughput testing which has increased the productivity of our lab. Chromatographic separation and higher-resolution ESI-TOF mass spectrometry has been advantageous as the increased resolution affords the ability to track M-proteins in serum to levels comparable to bone marrow based minimal residual disease detection. These advantages result in better care of patients with monoclonal gammopathies.

Methods:
Immunoaffinity purification with MALDI-TOF MS: 10 mcL of serum was added to 50 mcL of a 10% v/v slurry of CaptureSelect resin targeting each IgG, IgA, IgM, kappa or lambda. After a 15 minute sample incubation, 3 50 mcL PBS washes and 3 50 mcL water washes were performed. Isolated immunoglobulins were eluted with 30 mcL of 20 mM TCEP in 0.1% TFA with a 15 minute incubation for reduction. The isolates were spotted with 10 mg/mL CHCA matrix in 50% ACN with 0.1% TFA and acquired on a MALDI TOF mass spectrometer (microflex smart LS, Bruker) in positive ion mode, with spectra collection from 6000-32000 m/z. Spectra were reviewed using in-house developed software.

Immunoaffinity purification with high resolution ESI-TOF MS: 30 mcL of serum was added to 200 mcL of a 10% v/v slurry of CaptureSelect resin targeting each IgG, IgA, IgM, kappa or lambda. After sample incubation for 15 minutes, resin was washed 3 times with 500 mcL water. Isolated immunoglobulins were eluted with 100 mcL of 5% acetic acid, followed by reduction with 50 mcL DTT in 1M ammonium bicarbonate and incubated at 55°C for 30 minutes. Isolates were analyzed via an Eksigent Ekspert 200 microLC (Dublin, CA) for separation; mobile phase A was water + 1% FA, and mobile phase B was 80% acetonitrile + 10% 2-propanol + 0.1% FA. A 7 μL injection was made onto a 1.0 × 75 mm Poroshell 300SB C3, 5 μm column flowing at 25 μL/min. A 15 min gradient from 25%B to 50% B was used for immunoglobulin elution. Spectra were collected on an Sciex TripleTOF 5600 quadrupole time-of-flight mass spectrometer (Sciex, Vaughan, ON, Canada) in ESI positive mode with a Turbo V dual-ion source with an automated calibrant delivery system (CDS). Source conditions were IS, 5500; temp, 500; CUR, 45; GS1, 35; GS2, 30; and CE, 50 ± 5. TOF MS scans were acquired from m/z 600−2500 with an acquisition time of 200 ms. The instrument was calibrated every ten injections through the CDS using calibration solution supplied by the manufacturer. Data analysis was performed using Analyst TF v1.6 and PeakView version 2.2. The mass spectra of the multiply charged light-chain ions were deconvoluted to accurate molecular mass using Bio Tool Kit version 2.2 plug-in software. Deconvoluted mass spectra were reviewed manually.

Results:
Analytical comparison between mass spectrometry methods: For an IgG kappa monoclonal protein spiked at decreasing concentrations into normal human serum, the limit of detection measured by MALDI-TOF MS was 50 mcg/mL, and by ESI-TOS MS was 3.13 mcg/mL. Current gel-based limits of detection range from 20-200 mcg/mL. In a study spiking tmAbs into samples with endogenous IgG kappa clones, MALDI-TOF MS was able to resolve 87% of endogenous M-proteins from tmAbs, while ESI-TOF MS was able to resolve 100% of clones (Kohlhagen. Clin BioChem 2021 Jun: 92:61-66).

Light chain N-glycosylation: In a study from 2020, among 414 MGUS patients, 25 (6%) displayed N-glycosylated light chains and were found to have a higher likelihood of progression to AL amyloidosis over time (hazard ratio =10.1, 95% CI 2.9,34.7) (Dispenzieri. Leukemia 2020 Oct; 34 (10): 2749-53). N-glycosylated light chains were also observed in a high number of patients with cold agglutinin disease (N=9/14, 64%) compared to other IgM related gammopathies (N=31/438, 7%) (Sidana. Am J Hematol 2020 Sep: 95(9):E222-5). In an additional cross-sectional study of 6315 patients, N-glycosylation was observed at higher rates in patients with LC amyloidosis and cold agglutinin disease than in other disease groups (Mellors. Blood Cancer J 2021 Mar; 11(3):50).

Minimal residual disease: For 251/431 patients enrolled in the STAMINA trial having MRD bone marrow testing by high sensitivity flow cytometry at 1 year post induction, serum Mass-Fix negativity and MRD negativity predicted better progression free survival and overall survival (median follow-up time was 6 years with overall survival of 76%), while serum immunofixation negativity and complete response did not (Dispenzieri. Blood Cancer J 2022 Feb; 12(2): 27).

Conclusion: Mass spectrometry has advanced the way we detect and monitor M-proteins in the clinical laboratory. Observation of post translational modifications such as N-glycosylation of light chains and more sensitive detection of M-proteins already has had a positive impact on the care of patients with monoclonal gammopathies.

Unique Chemoselective Probes for Discovery and Investigation of Metabolites in Human Samples with Enhanced Mass Spectrometric Sensitivity Weifeng Lin (Presenter) Uppsala University

To be presented in Track 2 (Steinbeck 2) on Thursday at 14:20

Introduction: Metabolites produced by the gut microbiome play a crucial and diverse role on host physiology, which are detectable in a wide range of biological samples including feces, plasma, urine, and cerebrospinal fluid. Microbiota dysbiosis has been associated with the development of diseases, however, the metabolic link has yet to be detected. The detailed and targeted analysis of these metabolites is important for the discovery of biomarkers and unknown bioactive molecules.[1]

Methods: Mass spectrometric metabolomics is the method of choice for identification and quantification of these metabolites. Advanced methods at the interface of chemistry and biology coupled with metabolomics analysis are required but still limited. We have therefore developed a unique and multifunctional chemoselective probe with synthetic 13C/12C isotopically labelled analogues that allows for comparative and quantitative analysis of metabolites in human samples at low concentrations.[2] We have termed this unique method quantitative Quantitative Sensitive CHEmoselective MetAbolomics (quant-SCHEMA). Coupled to magnetic beads, this method allows the straightforward chemoselective extraction of metabolites from human samples.[2-6] The captured metabolites are released under mild conditions through the cleavage of the newly developed bioorthogonal cleavage site p-nitrocinnamyloxycarbonyl (Noc). This isolation procedure of specific classes of metabolites from sample matrices led to significantly increased mass spectrometric sensitivity by sixth orders of magnitude and facilitates the detection of metabolites at femtomole quantities.

Results: Herein, we have developed a new chemical biology tool using chemoselective modification to overcome analytical limitations. Due to the combination of natural and isotope-labeled conjugates before LC-MS analysis, this new method does not require any normalization procedure including internal standards or normalization of different sample types. This is necessary in general metabolomics analysis to reduce technical errors. Two isotopic probes allow for the simultaneous and semi-quantitative analysis at the femtomole level as well as qualitative analysis at attomole quantities that allows for detection of more than 200 metabolites in human fecal, urine and plasma samples. This method also facilitates detection of 21 metabolites that were not previously detected and can now even be quantified.It enhances the scope of metabolomics-driven biomarker discovery. We anticipate that our chemical biology tool will be of general use in metabolomics analysis to obtain a better understanding of microbial interactions with the human host and disease development.

Conclusion: The chemoselective probe was applied for analysis of amine- or carbonyl-containing metabolites in human fecal, urine and plasma samples. We have discovered several metabolites previously unreported and performed metabolic profiling in these sample types and confirmation of the presence of medically relevant gut microbiota-derived metabolites.

Reference:
[1] M.S. Donia, M.A. Fischbach, Science, 2015, 349, 1254766.
[2] W. Lin, L.P. Conway, M. Vujasinovic, J.-M. L&ouml;hr, D. Globisch, Angew. Chem. Int. Ed. 2021, 60, 23232-23240.
[3] N. Garg, L. P. Conway, C. Ballet, M. S. P. Correia, F. Olsson, M. Vujasinovic, J. M. L&ouml;hr, D. Globisch, Angew. Chem., Int. Ed., 2018, 57, 13805&ndash;13809
[4] L. P. Conway, N. Garg, W. Lin, M. Vujasinovic, J.-M. L&ouml;hr, D. Globisch, Chem. Comm. 2019, 55, 9080&ndash;9083.
[5] W. Lin, L. P. Conway, A. Block, G. Sommi, M. Vujasinovic, J.-M. L&ouml;hr, D. Globisch, Analyst, 2020, 145, 3822&ndash;3831.
[6] W. Lin, Z. Yang, A. Kaur, A. Block, M. Vujasinovic, J.-M. L&ouml;hr, D. Globisch, RSC Chem. Biol. 2021, 2, 1479-1483.

Evaluation of Mass Spectrometry Methods for Glycosaminoglycan Biomarker Quantification in Mucopolysaccharidosis and GM1 Gangliosidosis Newborn Dried Blood Spots Zackary Herbst (Presenter) University of Washington

To be presented in Track 1 (Steinbeck 1) on Thursday at 15:35

INTRODUCTION: Measurement of enzymatic activity in newborn dried blood spots (DBS) is the preferred first-tier method in newborn screening (NBS) for the mucopolysaccharidoses (MPSs). However, false positives are observed due mainly to the presence of pseudodeficiencies. Recent research has shown that second-tier measurement of glycosaminoglycan (GAG) biomarker levels in DBS for the MPSs can dramatically reduce the false positive rate in NBS. Additionally, these methods are useful tools in monitoring progression and treatment of MPSs and GM1 gangliosidosis, another Lysosomal Storage Disorder (LSD) which warrants GAG analysis.

OBJECTIVES: Two methods for measuring GAG biomarkers in 10 MPS conditions and GM1 gangliosidosis are directly compared in the analysis of DBS from newborns with positive diagnoses.

METHODS: MPS and GM1 gangliosidosis-diagnosed newborn DBS samples are analyzed against a reference range of healthy newborn DBS via two GAG mass spectrometry methods: (1) the internal disaccharide biomarker method, and (2) the endogenous biomarker method. The internal disaccharide method is the classical method for GAG analysis wherein GAG polymers in patient DBS are cleaved by bacterial lyases and hydrolases to yield many copies of a set of disaccharides from the repeating unit of GAG polymers. These disaccharides are then quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A second method, the endogenous biomarker method, analyzes the non-reducing end (NRE) of the GAG polymers without in vitro enzymatic digestion. Rather, endogenous NRE fragments generated by endohydrolases and exohydrolases in the human donor are derivatized and quantified directly by LC-MS/MS. A third NRE method, called the SensiPro method, involves enzymatic depolymerization of the GAG polymer, derivatization of the resulting glycans with aniline, and selective detection of the NRE derivative by LC-MS/MS. Due to similarities between the SensiPro method and the Internal Disaccharide method, in addition to complex sample workup requirements, the SensiPro method was only studied for MPS-I newborns.

RESULTS: The minimum differential factor (lowest GAG marker level in patient samples divided by highest level in a reference range of random newborns) was determined for both biomarker methods tested. The internal disaccharide minimum differentials for MPS-I and MPS-II were 2.5-fold and 3.0-fold, for MPS-IIIA and -IIIB were 3.7-fold and 5.4-fold, for MPS-IIIC and -VI were 9.3-fold and 2.1-fold, and for MPS-VII was 4.2-fold. The endogenous biomarker method minimum differentials for MPS-I and MPS-II were 16.0-fold and 3.8-fold, for MPS-IIIA and -IIIC were 4.6-fold and 3.2-fold, for MPS-IVA and-VI were 20.0-fold and 20.6-fold, and for MPS-VII and GM1 gangliosidosis were 10.0-fold and 3.0-fold, respectively. Non-newborn MPS-IIID DBS were tested for MPS-IIID against a reference range of healthy non-newborn DBS. The minimum differentials for these MPS-IIID DBS were 2.3-fold for the internal disaccharide method and 14.7-fold for the endogenous biomarker method.

CONCLUSION: Overall, the endogenous biomarker method outperforms the internal disaccharide method with regard to low variability in the reference range. While the internal disaccharide biomarkers give significant peak signals in healthy newborn DBS, the peak signals for endogenous biomarkers in healthy newborn DBS are either background noise or an order of magnitude lower than signals in patient DBS. Based on minimum differentials, the internal disaccharide method is the preferred method for MPS-IIIB and -IIIC but it is not able to discriminate MPS-IVA or GM1 gangliosidosis newborns from the reference range. The endogenous biomarker method is the preferred method for MPS-I, MPS-II, MPS-IIIA, -IIID, -IVA, -VI, -VII, and GM1 gangliosidosis and is generally the most useful method, with the exception that no endogenous marker was identified for MPS-IIIB DBS. MPS-IVB newborn DBS were not tested. This study supports the use of second-tier GAG analysis of newborn DBS, especially the endogenous biomarker method, as part of NBS to reduce the false positive rate.

Expanding the Tissue-based N-glycan Imaging MS Workflow into a Platform Technology for Glycan Targeted Biofluid and Cellular Diagnostics Richard Drake (Presenter) Medical University of South Carolina

To be presented in Track 2 (Steinbeck 2) on Thursday at 16:50

INTRODUCTION: Alterations in the glycosylation of circulating and cell surface glycoproteins is a hallmark of cancer, inflammation and most disease processes. High throughput and reproducible analysis of glycosylation in liquid biopsy and tissue samples at the clinical diagnostic level has long been challenging, due to lengthy processing, derivatization and processing workflows. Our group has collectively adapted an N-glycan MALDI imaging mass spectrometry method to multiple approaches for analysis of biofluids, cultured cells and immune cells using direct slide-based capture approaches.

OBJECTIVES: Develop a platform technology to rapidly and reproducibly detect glycan changes by MALDI-QTOF MS profiling in liquid biopsy clinical specimens.

METHODS: Serum cohorts related to early detection of breast and liver cancers were evaluated, as well as human and mouse peripheral blood mononuclear cells (PBMC). For biofluid glycan profiling, diluted aliquots of biofluids are spotted directly on amine reactive glass slides, rinsed, and sprayed (HTX imaging, Durham, NC) with a molecular coating of PNGase F to release N-glycans. For antibody array capture, antibodies to major serum glycoproteins and immune cell markers are spotted on amine reactive slides, and incubated with serum or immune cell samples. Released N-glycans are detected using a timsTOF fleX MALDI-QTOF (Bruker, Billerica, MA). Data analysis is done using SCiLS Lab software (Bruker, Billerica, MA)

RESULTS: The standard N-glycan MALDI IMS workflow for analysis of clinical FFPE tissues involves an antigen retrieval step followed by molecular spraying of PNGase F to release N-glycans. After application of matrix, the N-glycans are detected by MALDI MS. The enzyme spraying and detection steps have proven to be robust and reproducible across thousands of tissue samples. We have subsequently found that applying any biological sample that contains N-glycans to a slide can be assayed with essentially the same digestion and detection workflow developed for tissue slides. Four main approaches have been adapted to on-slide N-glycan analysis of non-tissue samples: cultured cells grown directly on slides; blood and urine spotted directly on amine-reactive slides; individual glycoproteins captured on antibody arrays; and individual immune cell types captured by anti-CD marker antibody arrays. The cultured cell and direct biofluid methods are established and published. Profiling of total N-glycan content in large cohorts of breast cancer and benign serum samples (n = 298), and liver cancer and cirrhosis serum cohorts, have been recently completed Analysis of N-glycans in lupus nephritis plasma, urine, and urine exosomes are ongoing. Using specific antibodies spotted onto slides to capture and N-glycan profile individual serum glycoproteins has been developed initially by targeting immunoglobulin G (IgG) and IgG sub-types. Serum or plasma samples are incubated in wells containing the specific antibody, and following rinsing, N-glycans attached to the captured glycoprotein are released by sprayed PNGase F and detected at each antibody-antigen spot by MALDI MS. Termed GlycoTyper, this approach is being optimized for commercial diagnostic applications for early detection of fatty liver disease, cirrhosis and liver cancers. In an ongoing approach using a serum cohort (n =200) representing subjects with cirrhosis or early liver cancers, a slide array that includes 15 additional antibodies to acute phase reactant serum glycoproteins was evaluated. The newest assay developed involves use of antibodies to immune cell markers like CD4 and CD8 to capture and glycoprofile individual immune cell subtypes directly on a glass slide. Different mouse and human immune cell mixtures have been used to optimize capture and processing details. A unique aspect of this assay is the visualization and counting of captured immune cells by light microscopy prior to glycan release and MS analysis. For each of the four assay formats, example applications and initial results in clinical samples, experimental parameter optimizations, data analysis and quality control issues will be discussed.

CONCLUSIONS: The method workflow originally created for N-glycan tissue imaging MS has been adapted to be an extensible and rapid glycan analysis platform. Glycoproteins in clinical biofluids and cellular samples captured on antibody array slides can be efficiently evaluated for N-glycan content using a timsTOF fleX MALDI QTOF. This clinical biospecimen glycan analysis platform technology is amenable to analysis of large clinical sample cohorts and can be significantly expanded to include additional antibodies of interest for glycoprotein and cell capture applications.

Combined Single Neuron Patch-Clamp/Mass Spectrometry (PatchC-MS) Analyses John Yates (Presenter) Scripps Research Institute

To be presented in Track 2 (Steinbeck 2) on Thursday at 17:10

As interest in single-cell analysis increases, performing single cell MS still remains a challenge. Herein we demonstrate patch-clamp electrophysiological recordings of single human iPSC-derived neurons followed by mass spectrometry analysis of the same cell. Human induced pluripotent stem cell (hiPSC)-derived cerebrocortical neurons are evaluated electrophysiologically by whole-cell recordings with a patch electrode capillary. The neuron is then aspirated into the capillary and expelled into a microtube. A simple digestion protocol is performed, and samples are analyzed by mass spectrometry. The single-cell digests are separated by nanoflow UPLC coupled to a Bruker timsTOF or a Thermo Eclipse, both operating in data dependent modes. Whole-cell recordings were performed on Alzheimer’s disease (AD) and isogenic, gene-corrected control (wild-type/WT) hiPSC-derived cerebrocortical neurons. WT neurons of interest were chosen based on their ability to fire action potentials, manifest voltage-gated sodium and potassium currents, and neurotransmitter-mediated postsynaptic currents. We have previously published that AD hiPSC neurons, like those in human AD brain, exhibit enhanced spontaneous action potential frequency, increased voltage gated sodium currents, and increased excitatory postsynaptic current frequency compared to WT neurons (Ghatak et al., eLIFE, 2019). We selected these AD neurons to compare to WT controls for further proteomic analysis. MS data analysis was performed with ProLuCID, Byonic and MSFragger. When injecting half of the contents of a single digested neuron, we were able to identify between 400-2000 proteins per sample. Advances in this methodology are used to perform patch clamping and proteomics analysis on neurons from brain tissue slices. We performed single-cell patch-clamp electrophysiology combined with mass spectrometry proteomic analysis.

Development of Thread Spray Mass Spectrometry for the Identification of Pulmonary Exacerbation Biomarkers in Cystic Fibrosis Salmika Wairegi (Presenter) The Ohio State University

To be presented in Track 3 (Steinbeck 3) on Thursday at 16:30

INTRODUCTION: This presentation focuses on the development of a non-invasive surveillance test based on an innovative skin patch for collection of sweat metabolites, which provides a convenient and easy-to-use platform for monitoring patients with cystic fibrosis (CF). CF is an autosomal recessive respiratory disease caused by mutation in the cystic fibrosis transmembrane regulator (CFTR) gene. The CFTR gene codes for the CFTR protein, which is an ion channel that controls the movement of chloride ions in and out of epithelial cells. Individuals affected with CF tend to experience periods of worsened lung function, which is referred to as pulmonary exacerbations (PE). In the event of PE, there is an acute worsening of lung function, which can lead to reduced responses to treatment. It is critical to effectively manage and prevent the onset of PE. Metabolites from various biofluids (e.g., blood and sputum) have been recognized as potential biomarkers for the management of PE. While useful, these biological sources can limited due to lack of ease of access at all ages. The proposed skin patch will collect sweat, which is a non-invasive biofluid that is easily accessed regardless of age. The absorbent in the patch is created from cellulose threads, which themselves provide a means by which the collected metabolites can be analyzed directly without prior sample preparation. This presentation will discuss the (i) creation of the skin patch, (ii) development of a novel solvent system that allows detection of both acidic and basic metabolites in a single experiment, and (iii) direct thread spray ambient ionization mass spectrometry method for metabolite characterization.

OBJECTIVE: The objective of this study is to develop a thread-based skin patch for prediction of PE in patients with chronic cystic fibrosis. Patients will wear the patch for a minimum of 24 h, after which the used patch will be mailed to us for subsequent analysis. We plan to detect the 5 selected metabolites in sweat samples directly from the thread substrate. To determine the efficacy of the selected metabolites as predictive biomarkers for the onset of PE, we will take samples before and after treatment. Data will be statistically analyzed by comparing the predictive power of the individual molecules versus the combination. We will determine the least number of metabolites that can provide the highest combined predictive effect.

METHOD: The patch will be created similarly to bandage where cellulose fabric of individual cellulose threads (inner layer) will be placed on an adhesive release liner. The assembly of the cellulose material and the adhesive tape will in turn be placed on an outer base layer (e.g., polyurethane adhesive). The inner cellulose layer will absorb the sweat from the skin once the patch is placed on the body (e.g., on the shoulder or forehead). The specific cellulose absorption material will be optimized by changing the thread density. When possible, the cellulose material will be pre-treated to remove endogenous chemicals that might be present in the commercial fabric. Individual cellulose threads in which the sweat has been absorbed will be removed from the patch and inserted into a glass capillary. The application of appropriate solvent enables selective extraction of interested analytes from the sweat sample. In this project, we employed direct thread spray ionization by adding 4 M NH4OH to the spray solvent MeOH:H2O (80:20, v:v) and applied a 5kV voltage. Method optimization was achieved artificial sweat where we spiked 200 μM of each metabolite individually. Structural characterization was performed using collision-induced dissociation in tandem MS (MS/MS). About 10 μL of artificial sweat was used in all preliminary studies.

RESULTS: The current work is a collaboration between our laboratory and Nationwide Children’s Hospital (NCH) in Columbus, OH. A previous study using liquid chromatographic mass spectrometry analysis of sweat collected with a commercial Wecor Macroduct® Sweat Collection System revealed the following 5 metabolites to hold the most potential as predictive biomarkers for the onset of PE before and after antibiotic treatment: 2-piperidone, thymidine, adipic acid, succinic acid and 2-hydroxy-3-methylbutyric acid. The goal of the current work was to translate this result into a clinical device for non-invasive surveillance of CF patients. The first step in this process was to evaluate the possibility of directly analyzing the sweat from the thread absorbing substrate. This was achieved in two separate experiments: (1) direct ionization of all metabolites from a single thread using a single spray solvent. We identified 4 M NH4OH in MeOH:H2O (80:20, v:v) to be effective at ionization both the acidic and basic compounds through a wrong-way ionization mechanism. The details of this new spray system and its implementation in thread spray MS will be discussed. (2) We performed MS/MS to determine diagnostic fragment ions that can be used to identify each metabolite in the presence of others. Based on these diagnostic ions, we will discuss sensitivity and accuracy of the method in this presentation. We have used the skin patch to collect from healthy volunteers and recorded comparable ion profiles for sweat obtained using Macroduct® from the same two people. This result and those obtained from CF patients will discussed in detail.

CONCLUSIONS: These results suggest that thread substrate is a suitable medium for collection sweat. Thread spray mass spectrometry provided an effective method for analysis of raw sweat directly from the thread substrate. Sample is found to be stable for at least 90 days after collection.

Towards Automated Point-of-Care Profiling of the Vaginal Microbiome Using Direct Swab Analysis by Desorption Electrospray Ionisation Mass Spectrometry (DESI-MS) Eftychios Manoli (Presenter) Imperial College London

To be presented in Track 4 (Colton) on Thursday at 16:30

INTRODUCTION

A sub-optimal vaginal microbiome is characterised by depletion of commensal Lactobacillus species and increased bacterial diversity. This is associated with common infections, like bacterial vaginosis (BV) and increased risk of developing sexually transmitted infections (e.g. HIV), gynaecological cancers, and in pregnancy, suffering miscarriage and preterm birth. However, a sub-optimal vaginal microbiome is often observed in asymptomatic women. Currently, vaginal infection is diagnosed by sending a vaginal swab to a laboratory for culture or microscopy testing. These tests are slow, inaccurate, and require highly trained experts to perform them. We have addressed these issues by developing Direct Swab Analysis by Desorption Electrospray Ionisation Mass Spectrometry (DESI-MS)1. This method works by measuring specific chemicals directly from the surface of routinely collected clinical swabs which can then be used to predict the microbiome composition and immune status of the sample within 2 minutes. The method was originally developed on a large-footprint instrument using an in-house, custom-built swab holder that limited the rotation of the swab to a fixed position. To facilitate the use of the method as a point-of-care device, we aim to transition the technique towards a smaller format, portable instrument and test the stability, reproducibility, and robustness needed to meet regulatory requirements.

OBJECTIVES

To transition our existing DESI-MS prototype from an LTQ Orbitrap mass analyser to a small footprint and robust time of flight (e.g. ACQUITY RDa Detector) instrument and perform analytical validation and assessment of technical variation introduced by sample collection and storage conditions, laboratory procedures or instrument performance.

METHODS

Vaginal swab samples were collected as part of a prospective observational cohort study from the early pregnancy clinic at Hammersmith Hospital, Imperial NHS Trust, London, UK. Swabs for DESI-MS, metataxonomic profiling of the vaginal microbiota, and immune profiling were immediately stored at &minus;80&thinsp;&deg;C upon collection. An additional swab was collected for Gram staining and microscopic assessment. DESI-MS data were acquired using the RDa/BioAccord mass spectrometry platform (Waters Cor., UK). For swab analysis and as part of the DESI stage, a roboticized sampling device was used. Data were acquired in both positive and negative ion modes in the m/z range of 50-2000. For data acquisition parameters the capillary voltage was set to 0.8-1.2 kV, gas pressure was set at 4 bar, source temperature at 120&deg;C, and gas desolvation at 450&deg;C. Swabs were placed into a rotating holder positioned orthogonally in front of the MS inlet capillary with a swab&ndash;capillary distance of approximately 2&thinsp;mm. The DESI sprayer tip was pointed to the swab centre with a tip-sample distance of 1.5&ndash;2&thinsp;mm and a distance between the tip and the inlet capillary of 2&thinsp;mm. The entire surface of the medical swabs was analysed by DESI-MS through clockwise rotation of the swab toward the MS capillary. A mixture of methanol/water (95:5, v/v) was used to produce the charged droplets and the desorption of analytes. Statistical and multivariate analysis was done in R and Python.

RESULTS

A swab holder capable of executing screw motion and simultaneous linear plane movement was designed and manufactured. The holder could be fitted onto the existing ionisation source (DESI, Waters RDa system), and seamlessly integrated with the current workflow. The device includes a magnetic, adjustable swab grip that facilitates the analysis of different swab types with differing diameters and lengths. Optimisation was achieved for several DESI geometric parameters and ergonomics, including distance of the swab tip from the swab-inlet capillary-solvent sprayer, facility of cleaning minimising contamination, rotation and linear speed and pause functionalities.

A quality control (QC) standard that provides a sample matrix mimicking the physical, biochemical, and microbiological properties of mucosal smears was developed and used to assess the instrumental stability and correction for drifts in signal intensity commonly observed during the analysis of samples. Twenty endogenous metabolites across the whole m/z range were identified and used to assess instrument performance.

Analysis of vaginal swab samples (n=20) by DESI-MS performed on an Orbitrap mass analyser or the ACQUITY RDa Detector showed comparable data including various small metabolites, polyunsaturated fatty acids, cholesterol sulphate and glycerophosphoserines. Differences in DESI-MS profiles acquired using the ACQUITY RDa Detector associated with vaginal microbial composition could be readily detected including differences in the relative abundances of oxypurinol and various lysophospholipids (p&lt;0.05).

CONCLUSIONS

We have developed a robust, easy-to-use, high-throughput, automated swab-holding device that allows rapid metabolic profiling of vaginal swabs in less than two minutes. Incorporation of QC&rsquo;s within the analytical workflow allows accurate and reproducible monitoring of instrument stability and performance. This integrated &ldquo;all in one&rdquo; benchtop vaginal swab analysis platform is currently being used in an ongoing large-scale analysis of vaginal swabs collected from 1000 women attending early pregnancy clinics. Future work includes the installation of an instrument in the clinical environment for data acquisition and the development of prediction models for vaginal microbiome status.

1. Pruski, P., Correia, G.D.S., Lewis, H.V. et al. Direct on-swab metabolic profiling of vaginal microbiome host interactions during pregnancy and preterm birth. Nat Commun 12, 5967 (2021).

Advanced Metabolomics Analysis Through Chemical Biology Tools for the Selective Investigation of Gut Microbiota-Derived Metabolites Daniel Globisch (Presenter) Uppsala University

To be presented in Track 4 (Colton) on Thursday at 17:10

INTRODUCTION: The detailed investigation of metabolites in human samples (serum, plasma, urine, saliva, feces or tissues), termed metabolomics, carries a great potential for the discovery of unknown biomarkers. The two major phase II modifications sulfation and glucuronidation have been linked to microbiota-human host co-metabolism.

OBJECTIVES: Our aim is the development of new methodologies to overcome limitations in sample preparation for subsequent mass spectrometric analysis. We are developing and utilizing techniques at the interface of chemistry and biology to allow for an advanced targeted qualitative and quantitative metabolomics analysis for the discovery of disease-specific biomarkers.

METHODS: We have developed a unique chemical biological technique by combining selective enzymatic metabolite conversion with metabolomics data analysis. We are selectively converting sulfated [1,2] and glucuronidated [3] metabolites in human urine samples for subsequent UPLC-MS/MS analysis using purified sulfatases and glucuronidases, respectively. The obtained raw data were then processed with R using the XCMS metabolomics framework to selectively identify metabolites with a sulfate and glucuronide moiety. We have performed MS/MS fragmentation for all identified features in order to validate the molecular structure. Recently, we have applied this methodology to a dietary intervention study [4]. To enhance the scope of our method, we have now applied two recombinant enzymes, ASPC and BG-Turbo, in order to increase the number of sulfated and glucuronidated metabolites and enhance the analysis for identification of additional scaffolds.

RESULTS: Through this advanced metabolomics analysis, we were able to detect 206 sulfated metabolites [1], which was three times higher than reported in the Human Metabolome Database at the time. Our approach for investigation of glucuronidated metabolites resulted in the detection of 191 metabolites including microbiota-derived compounds as well as previously unidentified molecules [3]. We have now obtained enzymatic hydrolysis profiles of sulfates and glucuronides with diverse scaffolds for the new recombinant enzymes.

CONCLUSIONS: We have succeeded in identifying numerous metabolites that have been correlated with microbiome host co-metabolism using these two new enzymes demonstrating the broad applicability of our analytical tool for elucidation of the cross-talk of gut microbes and their host.

REFERENCES
[1] C. Ballet, M.S.P. Correia, …, D. Globisch Chem. Sci. 2018, 9, 6233–6239.
[2] A. Jain, …, D. Globisch J. Pharm. Biomed. Anal. 2021, 195, 113818.
[3] M.S.P. Correia, M. Rao, C. Ballet, D. Globisch Chembiochem 2019, 20 (13), 1678–1683.
[4] M.S.P. Correia, A. Jain, …, A. Rodriguez-Mateos, D. Globisch Free Radic. Biol. Med. 2020, 160, 745–754.

Poster Presentations for Emerging Technologies

High-throughput Automated Evosep-MRM Workflow for Glycated Albumin Quantification in Diabetes Bharath Kumar Raghuraman (Presenter) Evosep Biosystems

Poster #2b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

INTRODUCTION: Glycated hemoglobulin (HbA1c) is the gold standard for the diagnosis of diabetes mellitus and for assessing glycemic status in these patients. Although HbA1c faithfully represents the average blood glucose level over the previous 8&ndash;12 weeks, its utility in monitoring the effect of medical interventions and diabetes progression is limited. Because HbA1c is prone to fluctuations in conditions like diabetic nephropathy, anemia, and pregnancy, glycated albumin (GA) has gained traction as a potential alternative due to its shorter half-life (2&ndash;3 weeks) and higher affinity to glycation.

OBJECTIVE: To develop a fully automated high-throughput Evosep-MRM workflow for quantifying GA in plasma/serum/dried blood spots.

METHODS: Five &micro;L of plasma was diluted in 295 &micro;L of 50 mM Tris-formate buffer (pH 7.5) in a 96-well plate. Diluted plasma (20 &micro;L~20 &micro;g) was digested using a modified solvent-assisted tryptic digestion (trypsin: protein-1:40) for 60 min and quenched using 4% formic acid before drying down. The complete sample handling was performed using an Opentrons OT-2 robot. The samples were reconstituted in 300 &micro;L 0.1% FA and 20 &micro;L was loaded to preconditioned Evotips using the OT-2 automated loading protocol and analyzed with Evosep One coupled to TSQ Altis using the standardized 300 samples/day or 500 samples/day method. Three intense transitions of a glycated and non-glycated form of KQTALVELVK peptide were monitored. The results were reported as %glycation of albumin (%GA).

RESULTS: The assay exhibited high repeatability and intermediate precision with a median coefficient of variation &lt;6% (intra-assay) and &lt;10% (inter-assay) across 3 QC levels of the JCCRM 611 GA calibration standard.

The stability of the glycated peptide in plasma and serum was explored for 3 handling temperatures (-80 deg C, +40 deg C, +220 deg C) for a span of 3 days. The %GA was stable at -80 deg C and +4 deg C for 3 days, whereas GA stability at room temperature was limited to 6 hours.
Further, we investigated the correlation between Evosep-MRM %GA and Tosoh G8 HPLC %HbA1c in random patient samples. The %GA and the %HbA1C exhibited a high, positive correlation, r=0.90. Next, the optimized assay was challenged with 407 patient samples with and without gestational diabetes mellitus to assess the robustness of the assay. Data will be presented.
We also further explored dried blood spots (DBS) as a sample source for the quantification of glycated albumin. Initial extraction protocols provided efficient solubilization of the proteins and the glycated albumin was quantified with a coefficient of variation of less than 10 %.

CONCLUSION: We have successfully developed a high throughput end-to-end automated assay for the quantification of glycated albumin in plasma, serum, and dried blood spots.

The Tipping Point: Lab Sustainability and the Future of Single-use Plastic Ali Safavi (Presenter) Grenova

Poster #7b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Description:
Operations of scientific laboratories are largely built upon the accepted use of disposable products. Every day, massive amounts of pipette tips, microplates, and cell flasks are consumed and thrown away after a single use, only to be incinerated or tossed into a landfill. In this session, we will discuss ways to minimize this detrimental impact on our environment by reducing the amount of waste generated from the assays conducted in labs, while maintaining the integrity of the data produced.

Background:
Disposable pipette tip usage for automated genomic assays is substantial, contributing to high operational costs and massive waste of plastic. Following one-time use, pipette tips are discarded as waste, incurring additional disposal costs. A solution to minimize the environmental impact is to implement innovative green technologies and manufacturing automated devices to wash and reuse plastic consumables for the laboratories. Studies have demonstrated the effectiveness of tip washing systems at removing molecular-level contaminants.

Discussion and Conclusion:
Our studies demonstrated the effectiveness of tip washing systems at removing molecular-level contaminants. Dirty pipette tips washed with a tip washer showed a 4-million-fold reduction in contaminating DNA compared to unwashed tips, which is sufficient to allow re-use for most applications. Additionally, control samples showed equivalent Ct values in fresh and washed tips, demonstrating that cleaning solutions used during the wash process are thoroughly removed by the rinse cycles, and that no interfering wear occurs. Tips come out of the washer in practically new condition and can be washed again and re-used up to 10 times, for a 90% reduction in tip costs.

High flow LC-MS analysis of plasma digest for protein identification Richard Gibson (Presenter) Thermo Fisher Scientific

Poster #8a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

Introduction: Human proteome analysis through liquid chromatography mass spectrometry (LC-MS) can play an important role in biomarker discovery in matrices such as plasma, serum or urine. Analyzing digested human plasma allows the detection of 1,000s of peptides within one run. Bioinformatic analysis then allows the identification of signature peptides that may be used to identify protein biomarkers of a disease. Data from high flow rate LC-MS has been shown to be able to identify 100s of proteins within one run that may then be used as targets for future studies.
MS2 scans can be data-dependent (DDA), data-independent (DIA) or targeted, such as with parallel reaction monitoring (PRM). Each of these scan types is accessible through high resolution accurate mass (HRAM) mass spectrometry (MS) and have been demonstrated to efficiently screen for proteins in plasma.

Methods: Plasma samples (heparin tubes) from a single donor obtained with IRB approval were centrifuged (800 RCF, 10 minutes). Plasma was mixed and transferred to 1.5 mL heparin tubes in 1 mL aliquots. An AB-1300 200 uL PCR plate is filled with 100 uL per well of 8 M GuHCl, 275 mM Tris-Hcl, 2% n-propanol, 10 mM DTT, pH 8.6. Plasma samples were vortexed and 30uL was added to each well. Samples were then mixed by pipetting. The plate was sealed using the Easypeel foil (AB-3739) on an APLS 3000 plate sealer and heated (37 oC, 1 hour). The seal was then removed, and 4.5 uL of 1M iodoacetic acid sodium salt (acros organics) was added to each well. The plate was then resealed, vortexed (1200 rpm, 5 minutes) and centrifuged (2500 RCF, 2 minutes). Nunc 2.2 mL deepwell plates (Nunc, 278752) were filled with 1.7 mL of 50 mM Tris-Hcl, 5 mM CaCl2. Samples were transferred to the deepwell plates containing 120uL of Pierce TPCK trypsin, 1mg/mL in 25 mM acetic acid in each well. The plates resealed and incubated (37 oC, 5 hours), after which 100 uL of acetic acid was added to each well, and the plates were resealed, vortexed and centrifuged (2500 RCF, 20 minutes).

From each sample 45 ug of digested plasma was injected into a VanquishTM Horizon UHPLC system fitted with a 2.1 x 50 mm PS-DVB trap column, and an AcclaimTM C18 120 Bonded phase 2.1 x 250 mm C18 2.2 um 120A pore analytical column with a 52 minute gradient and a flow rate of 250 uL/minute. The Orbitrap ExplorisTM 240 was set to a 50% AGC target value for the full scan with EASYIC. DDA, DIA and PRM data were collected in triplicate from various optimized MS2 scans. PRM precursor transitions were generated from a library created from DIA gas phase fractionation data.

Data was processed with Thermo Scientific Proteome DiscovererTM 3.0 and Skyline software.

Results: Protein and peptide IDs were determined from DDA, DIA and PRM scans. Precursor transitions for the PRM scans were chosen using seven small window gas phase fractionation (GPF) DIA scans. Selectively reducing the database size was demonstrated to increase the resolving power of the identification method, significantly increasing the number of protein and peptide IDs for DDA and PRM scans.

Conclusions: Protein and peptide IDs were calculated for DDA, DIA and PRM data, highlighting the versatility of HRAM mass spectrometry. Successful collection of DDA, DIA and PRM data highlights orbitrap technology being suitable for acquiring discovery data and translating it to a targeted method on the same instrument.

Automated Extraction and Quantification of Immunosuppressants from Dry Blood Spots Using the Transcend DSX-1 System Pragya Sharma (Presenter) Mayo Clinic

>> POSTER (PDF)

Poster #9b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Background:
Therapeutic drug monitoring (TDM) of immunosuppressants in whole blood is critical for clinical follow-up of transplant patients to obtain the optimum balance between therapeutic efficacy and the occurrence of adverse effects. Immunosuppressants are routinely quantified using LC-MS/MS in whole blood which translates to frequent phlebotomy visits for patients for the purpose of venous blood collection. Alternatively, dry blood spot (DBS) measurements have the potential to reduce the cost of sample collection and shipping, while simultaneously increasing the convenience of at home collection for patients. However, the analytical implications of DBS measurements must be carefully assessed to ensure the results are sufficient to fulfill the clinical need.

Objective:
To evaluate the analytical applicability of a DBS method for monitoring of immunosuppressants in venous blood samples using an automated extraction platform.

Methods:
Calibrators and QC were made by spiking immunosuppressants (Cyclosporine A, Tacrolimus, Sirolimus and Everolimus) into bovine whole blood. Residual venous blood samples previously analyzed for Cyclosporine A, Tacrolimus, Sirolimus and/or Everolimus on validated LC-MS/MS methods were utilized for this study. Calibrators, QC, and patient samples were spotted (25 μL) on PerkinElmer 226 Bioanalysis RUO Card with Ahlstrom 226 grade paper. Internal standards (IS) included Cyclosporin A-[15N]11 (Cerilliant), FK-506-[13C]-D2 (Cayman Chemical Company), Rapamycin D3 (Cambridge Isotope Labs Inc), and Everolimus-D4 (Cambridge Isotope Labs Inc). Immunosuppressants were extracted using a fully automated Thermo Scientific™ Transcend™ DSX-1 UHPLC system that performs internal standard addition, analyte extraction, 2-D LC matrix cleanup and analyte separation without any manual intervention. Sample analysis was performed on a Thermo Scientific™ TSQ Altis™ MD mass spectrometer and data was analyzed using TraceFinder™ LDT software.

Results:
Extraction, LC separation, and quantification of immunosuppressants using the Transcend™ DSX-1 system was accomplished using a 10.95-minute method. This method includes extensive wash and equilibration steps to minimize carryover. The intra-run imprecision of four different concentrations of QC for the four immunosuppressants measured was ±12% (n=9). The signal-to-noise ratio for all samples was greater than ten. Patient results from the Transcend™ DSX-1 were compared to established LC-MS/MS testing methods that utilized osmotic shock lysis by water followed by protein precipitation with methanolic zinc sulfate. Linear regression comparison indicated less than or equal to 10% bias and R² greater than 0.85 for all analytes tested.

Conclusion:
Preliminary studies suggest that the Transcend™ DSX-1 platform has the potential to provide a fully automated extraction of DBS for immunosuppressants. This sample matrix enables convenient sample collection for patients for therapeutic drug monitoring of immunosuppressants. Although our initial results are promising, additional studies are required to demonstrate if this platform can achieve the necessary analytical performance characteristics and determine if simultaneous venous whole blood collection and DBS of patients undergoing immunosuppressant therapy produce clinically equivalent results.

Simultaneous Detection of Respiratory Infectious Diseases Using Immunoprecipitation and LC-MSMS Yvonne Song (Presenter) Thermo Fisher Scientific

Poster #10b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

Introduction:

We are now living in a new era with new normal after the COVID-19 pandemic. Recently, respiratory syncytial virus (RSV) has been another concern as it surges among children. In addition to influenza viruses, not only are their symptoms similar at early stages, but they are also both enveloped viruses with several common biological properties, often leading to challenges in accurate identification. Thus, there is a need to develop a faster and more specific analytical tool that can differentiate infectious diseases.

Among different viral components, nucleocapsid protein or nucleoprotein (NP) is highly conserved and specific for infectious disease virus types. Therefore, targeting NP could provide a more robust and faster way for disease identification.

This study describes a targeted approach for the simultaneous detection of NPs from different respiratory infectious diseases using immunoprecipitation (IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Objectives:

To monitor multiple infectious diseases in a fast and sensitive way using immunoprecipitation and selected reaction monitoring.

Methods:

Prior to IP, equal amounts of all biotinylated antibodies were pooled together as one antibody panel for this study. The biotinylated antibody panel was added to samples collected via nasopharyngeal swabs in viral transport media (VTM) followed by incubation for 15 minutes at 25 C degree with rotation. The antigen-antibody complex in VTM was directly subjected to IP using Thermo Scientific™ Pierce™ MS-Compatible IP Kit (Streptavidin).

The IP purified samples were then digested for 15 minutes at 70 C degree with vortexing at 1000 rpm using SMART Digest™ Trypsin Kits and analyzed by Thermo Scientific™ Vanquish™ MD HPLC system hyphenated to Thermo Scientific™ TSQ Altis MD mass spectrometer. Data acquisition, processing and reporting were performed using TraceFinder™ LDT software 1.0.

Results:

Multiple viruses, SARS-CoV-2, influenza virus A and B types, RSV, and human coronavirus (HCoV-229E), were selected to show that this method can distinguish different disease viruses. The protein sequences of those selected NPs are mostly unique to each disease type and remain unchanged regardless of their variants, resulting in a reliable target peptides list for selected reaction monitoring (SRM). Combining with IP which uses a specific antigen-antibody interaction, it provides a highly targeted and confident detection of each disease. Thus, this approach is much simpler and robust with minimal changes when compared to other methods targeting different components such as the spike protein.

The workflow was optimized from sample preparation to LC-MS analysis. The protein precipitation and post sample clean-up were eliminated. From IP procedure, two incubation steps for antigen-antibody complex formation and immobilization on the magnetic beads were reduced to 15 minutes each (originally 1 hour each). Trypsin digestion incubation time was optimized to 15 minutes (previously 90 minutes). Particularly, the reduction of trypsin digestion time was achieved owing to a generation of much cleaner sample matrix by IP. The entire process was finalized to less than 1 hour from 4 hours. LC-MS run time was also optimized to 5 minutes.

A total of 12 peptides were successfully monitored (2 to 3 peptides per disease) by SRM. Calibration curve was generated with stable isotope-labeled standards (Thermo Scientific HeavyPeptide AQUA Ultimate). With criteria of % RSD < 15, CV < 15, and R2 > 0.99, LOQs were determined to be between 0.05 and 1 fmol of peptides on LC column with retention time variation ± 0.01 minutes. The method significantly improved the sensitivity and turn-around time, compared to other digestion or peptide enrichment methods.

Conclusion:

• Simple and robust approach by targeting the nucleoprotein component of the enveloped virus.
• Quick sample preparation taking < 1 hour and virus detection using 5-minute LC-MS method.
• MS-compatible and clean sample matrix generation by immunoprecipitation.
• Highly targeted, sensitive, and confident detection by immunoprecipitation and selected reaction monitoring.

For Research Use Only – Not For Diagnostic Procedures.

Simultaneous Determination of Different Classes of β-lactam Antibiotics in Human Plasma Shalini Varghese (Presenter) SCIEX

Poster #13a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
β-Lactam are among the most widely used class of drugs for the treatment of bacterial infections in humans. Most β-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organism. Bacteria often develop resistance to these antibiotics by synthesizing a β-lactamase, an enzyme that attacks the β-lactam ring common to this class of antibiotic. To overcome this resistance, β-lactam antibiotics can be given with β-lactamase inhibitors such as clavulanic acid. The antibacterial characteristics the drugs display are dependent on both the concentration of drug in relation to the minimum inhibitory concentration (MIC) and the time that this exposure is maintained. Therefore, monitoring their concentrations in plasma is of high importance.

In this study, a fast LC-MS/MS method with a simple sample preparation on the QTRAP 4500 system is described for the quantitative analysis of nine β-lactams antibiotics, amoxicillin (AMO), cloxacillin (CLO), piperacillin (PIP), cephazolin (CEP), cefotaxime (CEFO), ceftazidime (CEFT) and cefepime (CEFE), imipenem (IMI) and meropenem (MER).

Methods:
A 100 μL aliquot of each sample, calibrator or QC was spiked with 10 μL of internal standard mix at 100 μg/mL. Protein precipitation was carried out by the addition of 200 μL of methanol containing 0.1% formic acid. Following vortex mixing, the samples were centrifuged for 10 minutes before the supernatant was transferred to autosampler vials for injection. Chromatographic separation was accomplished using a Phenomenex Kinetex Biphenyl column (100 x 2.1 mm, 2.6 μm). Water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B) were used as mobile phase solvents. A 1 μL aliquot of the sample was injected into the UHPLC system. MS/MS detection was performed using the SCIEX QTRAP 4500 system equipped with Turbo V ion source using electrospray ionization, operating in positive mode. Multiple reaction monitoring (MRM) mode was employed, using two specific transitions of each analyte.

Results:
Calibration curves over the concentration ranges were between 1 and 150 μg/mL for all analytes except imipenem (0.05 to 7.5 μg/mL) and meropenem (0.1 to 15 μg/mL). Curves were generated using a 1/x weighted linear regression of the peak-area ratios of the antibiotic to corresponding internal standard. Regression coefficients of all calibration curves were greater than 0.99 with back-calculated concentrations of the calibration samples within ±15% (±20% at LLOQ) of the nominal concentrations. The recorded accuracy and precision were within European Medicines Agency guidelines. The application of this assay for nine β-lactams antibiotics was then used to analyze plasma samples from the Hospital of Mulhouse Antibiotic Therapeutic Drug Monitoring (TDM) program and compared to alternative methods for the analysis of these compounds.

Conclusions:
The QTRAP 4500 system was used for the quantitative analysis of nine β-lactams antibiotics, amoxicillin (AMO), cloxacillin (CLO), piperacillin (PIP), cephazolin (CEP), cefotaxime (CEFO), ceftazidime (CEFT) and cefepime (CEFE), imipenem (IMI) and meropenem (MER). Sensitivity was shown to be 1 μg/mL for all analytes except imipenem (0.05 μg/mL) and meropenem (0.1 μg/mL) in plasma.

PFAS in Me: Which Ones and How Much?! Karl Oetjen (Presenter) SCIEX

Poster #13b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Introduction:
Bioaccumulation of PFAS in the human body resulting from environmental exposure is a growing public health concern. Recent studies have linked PFAS exposure to adverse health outcomes including childhood health complications, reduction in kidney functions, thyroid disease, hormone suppression, decreased fertility, increased cholesterol levels, and diabetes, among others. Given the prevalence and ubiquitous nature of PFAS in the environment and every-day consumer products (including our drinking water supply), there is a critical need to develop quantitative tools capable of accurately and precisely detecting low-levels of PFAS in biological fluids in order to understand the impact on the human body.

In this study, we combined low volume blood sampling with the SCIEX QTRAP 7500 system for the analysis of trace level of PFAS. We present here a quantitative workflow capable of accurately quantifying sub-ng/mL levels of 42 PFAS compounds. The analysis was performed on the author and the results of the analysis are shared to demonstrate what PFAS exposure looks like in a typical American.

Methods:
A finger-prick was used to draw capillary blood. The first drop of blood was wiped away with a PFAS-free gauze and the Mitra device is applied to the subsequent drops of blood. The four Mitra tips contained approximately 30 µL of blood and were stored at -20°C until extraction. Absorptive Mitra tips were then removed from the stem and placed in polypropylene vials with isotopically labelled internal standards or IDAs (Isotope Dilution Analytes) and acetonitrile to aid with protein removal. The samples are sonicated and allowed to equilibrate prior to a centrifuge step to condense the precipitated protein for easier removal. The supernatant was removed and the original tube with Mitra was washed with solvent and the centrifuge step repeated to ensure that PFAS were not absorbed to the vial. The extracts were then combined and solid phase extraction (SPE) was performed. Injection internal standards (ISs, or recovery standards) were added to the SPE extract immediately prior to placing it in a new polypropylene vial for analysis.

These extracts were injected onto a C18 column at 30°C. A secondary column was introduced as a delay column to counteract endogenous interferences from environmental PFAS compounds present in the system. Data were collected using a SCIEX QTRAP 7500 system using electrospray ionization (ESI) in negative mode. The Scheduled MRM Algorithm was used to optimize data sampling across each peak and maximize the dwell times used.

Results:
The assay showed excellent analytical reproducibility, precision, accuracy, and linearity. The total amount of PFOA detected was 0.82 ng/mL, however, only the linear version of perfluorooctanoic acid (PFOA) was detected. This value was slightly lower than the median value of 0.9 ng/mL for Americans aged 18-49 according to the United States Environmental Protection Agency. The total amount of perfluorooctanesulfonic acid (PFOS) detected was 1.862 ng/mL, which again is lower than the median value listed by the EPA of 2.6 ng/mL. Finally, perfluorohexanesulfonic acid (PFHxS) was detected at a value of 1.558 ng/mL or 2.7 times higher than the listed median EPA value of 0.6 ng/mL.

Conclusions:
A robust and sensitive workflow for the detection of PFAS in blood samples using the SCIEX QTRAP 7500 system was successfully developed. This low-level sampling approach means that at-home testing of these compounds is possible and can help the population understand their PFAS exposure and the implications PFAS may have on their own health. While the concentrations of PFOA and PFOS presented in this study remained under national median values, the high concentration of PFHxS likely was related to past exposure to aqueous film-forming foam (AFFF).

Application of LC-MS/MS to Assess the Chemosensitivity of Breast Cancer Spheroids for an in vitro to in vivo Extrapolation Analysis Ramisa Fariha (Presenter) Brown University

Poster #15a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
While 2D in vitro cell culture models have been used over the years, 3D cell culture technique is becoming increasingly popular due to its ability to replicate the in vivo tumor environment better. Out of the existing 3D cell culture techniques, MicrotissuesTM has garnered attention due to its scaffold-free nature and high-throughput ability. However, traditional cell culture assessment techniques are unable to fully characterize this 3D platform due to limitations of quantifying trace amounts of absorption taking place within these molds.

Objectives:
Our study expands the use of LC-MS/MS to quantify the absorption of Paclitaxel, a chemotherapeutic, by the 3D MicrotissuesTM molds, and its subsequent impact on MCF7 breast cancer tissue viability.

Methods:
MicrotissuesTM molds were made using 2% agarose and MCF7 cells were allowed to self-assemble to tissue spheroids over DIV= 3. Drug adhesion was initially monitored for the empty wells and molds. Following the initial observation trend and drug absorption, the spheroids were treated with the actual concentration (measured after absorption by mold) versus the concentration it should have been receiving for 12 and 24-hours intervals to assess the impact of the minute change on cellular viability.

Results:
By optimizing the sample preparation technique, our strategy utilizes cell culture media to accurately quantify the drug adhesion to the MicrotissuesTM molds, with LoD= 0.03 μM, optimized for cellular microenvironment with relevance for extrapolation to patient application. The overall assay exhibits linearity of R2>0.99 inclusive of both MRMs, and a percent coefficient of variation that is less than or equal to 10%. We have tested and validated the findings against a traditional live-dead assay for the spheroids. In addition to manual plating with cell culture supernatant testing, we have exhibited the adaptability of our protocol on the JANUS G3 liquid handling workstation for rapid sample preparation, in addition to the fast three minutes total run time per sample analysis. This makes our protocol adaptable for clinical usage, expanding its application to personalized cancer therapies.

Conclusion:
Overall, our work plays an important role in the in vitro to in vivo extrapolation study of chemotherapeutics for breast cancer and how LC-MS/MS can provide a viable and highly sensitive and quantitative method for analysis.

Immunoprecipitation Top-Down High-Resolution Mass Spectrometry for the Quantification of the Protein Tumor Biomarker Neuron-Specific Enolase Sebastian van den Wildenberg (Presenter) Eindhoven University of Technology

>> POSTER (PDF)

Poster #18a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
LC-MS methods using bottom-up based and/or middle-down have been popular methods for the quantification of proteins. However, the development of these methods often come with extensive sample preparation that require thorough assay optimization. In addition to this using bottom-up proteomics sequence coverage is often limited and information about Post Translational Modification’s (PTMs) is lost. Using top-down proteomics intact protein are analyzed, without digestion, reducing sample preparation and retaining the maximum amount of information. Top-down proteomic approaches have their own specific challenges, such as limited sensitivity and the availability of internal standards and reference material.

Objectives:
The primary objective of this study is the development of an immunoprecipitation assay, followed by intact top-down protein analysis using LC-QToF-MS for the quantification of the small cell lung cancer (SCLC) tumor biomarker Neuron Specific Enolase (NSE).

Methods:
Immunoprecipitation was performed by coupling Protein-G-labeled magnetic Dynabeads™ to monoclonal antibodies against NSE. The protein-G-antibody complex was crosslinked with BS(PEG)5. Human (NSE-spiked) serum was incubated with antibody coupled beads. After incubation, the beads were collected using a magnet and serum was removed. Collected beads were washed and the captured NSE proteins were eluted from the beads. The eluted proteins were separated using RP-LC and analyzed by QToF-MS.

Results:
The immunoprecipitation method was developed and optimized for the isolation of NSE. Protein elution from the antibody-antigen-complex was conducted using water-acetonitrile (80:20) + 1% formic acid. Using this method recombinant NSE was successfully isolated from spiked human serum. Adequate linearity and sensitivity were achieved in the clinically relevant concentration range of 0 to 100 ng/mL using both QToF-Full Scan MS + Extracted Ion Chromatogram (XIC) and QToF-Selected Reaction Monitoring.

Conclusion and Outlook:
An immunoprecipitation method couple to a LC-QoF-MS method was developed and optimized for the isolation and quantification of recombinant NSE from spiked human serum. Next steps will focus on the isolation of endogenous human NSE from serum and optimization of the chromatography by transferring the method from UPLC to nano-LC. Additionally, QToF-MS- methods and data collection modes that can be used are compared, such as mass deconvolution, single reaction monitoring (precursor > product), pseudo-single reaction monitoring (precursor > precursor), extracted ion chromatograms and combinations.

Reduced Ion Suppression in an Automated Extraction of Vitamins B1 and B6 from Whole Blood for LCMS Analysis Kyle Dukes (Presenter) Biotage

Poster #24b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

Introduction:
Concentrations of Vitamin B1 (Thiamine Diphosphate, TDP) and B6 (Pyridoxal-5-Phosphate, PLP) in whole blood are measured clinically to help measure various metabolic functions. Accurate concentrations of vitamins B1 and B6 are difficult to obtain due to ion suppression found in LCMS analysis. Phospholipids and proteins are a main cause of the ion suppression. Currently, laboratories are struggling on creating accurate, precise and robust sample preparation methods focused on removal of these interferences.

Objectives:
The goal of this study is to provide a sample preparation method providing accurate and precise concentrations of vitamin B1 and B6 through the removal of interfering phospholipids and proteins. This study further aids in accuracy and precision by use of automation.

Methods:
The Biotage ISOLUTE® PLD+ protein and phospholipid removal plate is used in conjunction with the Biotage Extrahera sample preparation workstation to automate the extraction of vitamins B1 & B6 from whole blood. Vitamins B1 & B6 are highly water soluble (logP= -5.9, -2.2 respectively) and are extremely light-sensitive. An acidic, aqueous crash solvent was used for whole blood in order to extract the vitamins. MRM of three ions each were monitored for quantitation of vitamins B1 & B6. Phospholipid and protein removal is measured by monitoring the 184-product ion in precipitated samples compared to the samples prepared with ISOLUTE® PLD+.

Results:
Five replicate extractions were performed on whole blood samples which were precipitated alone and five samples which were precipitated with the ISOLUTE® PLD+ protein and phospholipid removal plate. Recoveries for vitamins B1 & B6 were 94% and 82% respectively with a %RSD < 2. Phospholipids and proteins were reduced by over 90%. Percent recovery is based upon the calculated recovery of the vitamins B1 & B6 by precipitation alone, highlighting any analyte loss due to the use of the ISOLUTE® PLD+ sample preparation plate.

Conclusion:
Matrix phospholipids and proteins were successfully removed (>90%) from whole blood vitamin B1 & B6 analysis using the ISOLUTE® PLD+ protein and phospholipid removal plate. Recoveries for vitamins B1 & B6 were 94% and 82% respectively with a %RSD < 2. Additional studies will be performed to determine if this method is suitable for other biological matrices as well.

A New Infographics-Guided Method Using Multimodal Imaging Mass Spectrometry Technology for Chemists, Pathologists and Surgeons Behnaz Akbari (Presenter) Boston University

>> POSTER (PDF)

Poster #25b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Introduction
Advanced mass spectrometry (MS) has broadly grown in tissue sample analysis, namely, imaging mass spectrometry (IMS), imaging mass cytometry (IMC), high-throughput analysis of tissue microarrays using automated desorption electrospray ionization mass spectrometry (DESI-MS), MasSpec pen, intelligent knife (iKnife), Laser Desorption Probes (Handheld probes), and topography molecular imaging with the assistance of a robotic arm coupled with water-assisted laser desorption (SpiderMass). However, intraoperative applications of MS require to be introduced as routine techniques for pathologists to improve patient healthcare and to extend the assessment of decision-making skills for surgeons.

Objectives
The bold ambition is to promote MS to the broader clinical community, integrating with quantitative multimodal imaging to visualize the data hidden in tissue samples. Because in a clinical setting, the primary concern is to analyze thin tissue sections from patients after sample preparation which could negatively affect sensitivity analysis of assurance methods and imaging quality. This is so we can link IMS and or IMC techniques to other imaging platforms, allowing interactions with pathologists, image analysis scientists, and surgeons in biomarker discovery, immuno-oncology, neuroscience, and metabolism.

Methods
Outline an IMS infographic, integrating imaging tools such as HALO and SciLS Lab, providing an understanding of quantitative multimodal imaging with simultaneous histological assessment.

Results
The infographic IMS project incorporated educational technology and engaged the multi-disciplinary team (chemists, pathologists, and surgeons) in clinical laboratories. Several beneficial features, such as a quick overview of IMS technology in the clinical setting and multimodal imaging integration, could promote visual literacy and develop creativity in the imaging domain.

Conclusion
The infographic IMS project helps clinicians understand the world of multimodal imaging workflows for later use at the operating suite housed in a hospital to aid pathologists and surgeons in exploring emerging questions in immunology, neuroscience, and cancer.

Optimization of Lead Recovery in Dried Blood Spot (DBS) During Method Development by ICP-MS Cody Orahoske (Presenter) University of California San Francisco

Poster #33a View Map

This poster will be presented and discussed on Wednesday at 12:00 for 15 minutes in De Anza 2 (right outside Exhibit Hall doors across from Ionpath in Booth 36).

1. Problem
In the development of this assay, we encountered insufficient extraction efficiency and irreproducible recovery.

2. Method Information
• Spot a Whatman 903 DBS card with 50 ul of whole blood, dry for at least 2 hours
• Cut a 6mm disc using a standard hole puncher
• Add DBS disc into a 15 ml conical tube
• Optimize extraction:
o Add 5 ml of extraction buffer into 15 ml conical tube; use extraction buffer with increasing EDTA concentrations, +/- additional compounds such as nitric acid
o Vortex for 1, 5, 15 and 30 minutes at various rpms
o Incubate for 30, 60 and 90 minutes
o Centrifuge for 5 minutes
• Run samples by ICP-MS, injection volume of 2.5 ml
• Interpret and analyze results for accuracy and imprecision

3. Troubleshooting steps
We hypothesized that there were five potential contributors for the insufficient lead extraction from filter paper: extraction buffer, vortexing speed, incubation time, the volume assumption of the punch DBS and the calibration curve. We systematically addressed each parameter to optimize the extraction conditions of lead from the DBS.

4.Outcome
The extraction method was optimized. The optimized method for a 6 mm disc from the filter paper card consists of using an extraction buffer containing 5mM EDTA and 0.05% triton, vortex speed of 1 minute at 2,000 RPM, and an assumption of 10 µl of blood from the DBS disc. When adding nitric acid to the extraction buffer we observed no dramatic improvement. Additionally, when increasing the vortexing time at 2,000 rpm we started to observe disintegration of the DBS disc. Importantly we found that using a DBS calibrator is necessary for accurate and reproducible recovery, relative to the use of a liquid calibrator in extraction buffer matrix. The DBS calibrator likely accounts for any matrix effect from the DBS card and corrects for loses during the extraction process. An incubation time of 30 minutes post vortexing is sufficient for maximum extraction with no apparent extraction increase after 90 minutes.

More Bang for Your Buck: Maximizing Sensitivity by Multifactorial Optimization of Method Parameters for Clinical Mass Spectrometry Evan McConnell (Presenter) Labcorp

Poster #35a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
Analytical sensitivity is a prerequisite for robust and reliable assays and is itself dependent on method parameters used by mass spectrometers (i.e., ion source and compound-dependent settings). Despite this importance, optimal settings are often considered de facto for a particular mode of instrument operation instead of being determined empirically. Care must be taken when developing new or transferring established assays onto alternate mass spectrometers as particular settings can be sub-optimal depending on the make, model, and application. Herein, we demonstrate an injection-based routine to screen method parameters on seemingly related mass spectrometers, noting key differences in optimal settings to achieve the best assay performance.

Methods:
Samples were analyzed by LC-MS/MS on an ARIA™ TLX-4 system (Thermo Scientific) coupled to either a Triple Quad™ 5000, 5500, or 7500 mass spectrometer (SCIEX). Both the 5000 and 5500 models used a Turbo V™ ion source equipped with an electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) probe. Similarly, the 7500 system used an OptiFlow™ Pro ion source configured to operate in either ESI or APCI mode, with the latter requiring the removal of the E Lens™ Technology.

Clinically-relevant assays utilizing selected reaction monitoring (SRM) were chosen as representatives for positive ESI (thyroglobulin – Tg), negative ESI (Reverse T3 – rT3), and positive APCI (Testosterone). The LC setup was different between assays; however, separation was generally performed using reversed-phase chromatography at ≥ 1 mL/min with a total cycle time ≤ 4 min.

The relative performance (i.e., peak area and signal-to-noise ratio) for SRM transitions specific to each analyte were compared across replicate injections of both reference standards and extracted specimens. Method parameters were screened using a custom AutoIt program to create acquisition methods with the desired range of values and step size for any combination of settings. Source variables considered were nebulizer gas (GS1), heater gas (GS2), curtain gas (CUR), collision gas (CAD), IonSpray™ voltage (ISV), and ion source temperature (TEM), with nebulizer current (NC) optimized for APCI only. Transition-specific variables included entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP). Additionally, declustering potential (DP) was ramped for 5000 and 5500 systems, while the analogous Q0 dissociation in simple mode (Q0DS) was specific only to 7500 models.

Results:
On 5000/5500 systems, positive ESI preferred high GS1 (≥ 40 psi) and ISV (≥ 4000 V) settings for the tryptic peptide specific for Tg protein. Negative ESI for the iodine-containing, small molecule rT3 was less dictated by GS1 (≥ 20 psi) and instead dependent on ISV (≤ -4000 V). Conversely, both ESI modes on the 7500 model were optimized with low settings for GS1 (10 to 40 psi) and ISV (positive: 1500 to 2500 V, negative: -1500 to -3000 V), which is attributed to the E Lens™ Technology improving desolvation by increasing the field strength experienced by ESI droplets. Testosterone by positive APCI mode had markedly similar optima for GS1 (≥ 50 psi) and NC (≥ 3 µA) on all systems.

Higher values for TEM generally increased analyte peak area, along with an especially noticeable increase in background noise on the 7500 system. GS2 is only used with ESI modes and produced minor differences on all systems regardless of the values tested (20 to 90 psi). As expected, increasing curtain gas decreased signal intensity for all analytes and primarily served as a protective barrier from contamination of the source and ion optics.

The CAD parameter specific to a method was ramped along with transition-specific CE to determine the best conditions for analyte fragmentation, which optimized to similar values regardless of the instrument model. Additionally, values for EP and CXP did not improve assay performance regardless of the settings used. Unique to the 7500 systems, Q0DS was found to attenuate signal intensity with increasing values (i.e., in-source fragmentation), similar to DP on 5000/5500 models. With the optimal Q0DS value enabled, both ESI modes demonstrated similar response compared to acquisition with this parameter disabled. Uniquely, positive APCI showed a four-fold enhancement in signal with Q0DS enabled (40 V), likely due to enhanced ion declustering.

Conclusion:
Injection-based optimization of method parameters used by mass spectrometers provided a simple means to ensure assay performance for routine clinical analysis.

The Development of a Virtual Liquid Chromatography Method Development Tool Melinda Urich (Presenter) Restek Corporation

Poster #38a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

INTRODUCTION: The development and optimization of liquid chromatography (LC) separations can be time consuming and costly, often requiring a number of steps including literature research, column selection, method scouting, method development, and method optimization. In an effort to eliminate these steps, an instrument-free, software modeling tool that gives users the ability to select compounds from a database and instantly model a separation on different column phases was developed. Optimization of the model can be performed while maintaining critical pair separations by adjusting for instrument/system effects (e.g. dwell volume and extra column volume), mobile phase preferences, number of gradient steps, and more. The modeler delivers a fast, no-cost starting point. The initial database consists of a Drugs of Abuse (DoA) library containing approximately 250 compounds with plans to continually expand the utility.

OBJECTIVES: To develop a chromatogram modeling tool that allows users to develop and optimize their LC methods virtually, improving data quality and laboratory efficiency without time-consuming in-lab method development.

METHOD: To build the chromatogram modeler, a DoA library containing approximately 250 compounds was created.

Retention times were first collected using a fast/slow gradient, 30°C/60°C temperature points, and ACN/MeOH mobile phases on a single column dimension. Some additional data points outside of these runs were also collected for the development of a semi-empirical correction factor that was used to improve modeling accuracy.

To assess the accuracy of the modeler, experiments comparing compound retention time values between wet-lab and modeled data were conducted. After the initial DoA library was built, the modeler was evaluated over four increasingly more complex stages of verification. In the final, most complex stage, new compounds not previously part of the initial DoA library were added and then compared by testing two different column dimensions, two different columns lengths, two different mobile phases, two different stationary phases, three different gradients programs, and three different temperatures against modeled retention time values. Because the semi-empirical correction factor was developed using only the original library compounds, this stage assessed the viability of adding future compounds to existing libraries.

RESULTS: An online chromatogram modeling tool was successfully developed that allows users to select columns and compounds for separation. A modeled chromatogram and instrument-ready conditions are automatically generated and can be further optimized by users. During software development, the acceptance criteria for retention time agreement between wet-lab and modeled values was set at +/- 15 seconds. This range was chosen because it represents a typical MRM window. In the most complex portion of the verification, 704 retention time data points were collected in total for the 25 compounds used in the evaluation. Only 13 data points exceeded the +/- 15 second window with no compounds missing acceptance criteria by more than five seconds, giving an overall pass rate of 98.2%.

CONCLUSION: For LC method developers, novice and expert, who either lack the expertise, or the time, to develop separations quickly and accurately, this free tool can be used to deliver a fast, no-cost starting point for method development and optimization. This novel, virtual method development software can improve turnaround time, increase throughput to existing methods, and offer an on-demand consultative user experience.

Miniaturization Strategies for Streamlined Drugs of Abuse Extraction Prior to UHPLC-MS/MS Analysis Lee Williams (Presenter) Biotage GB Limited

Poster #51b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Introduction
Miniaturized sample preparation, particularly solid phase extraction (SPE), has gained popularity in recent years with perceived benefits such as lower solvent consumption, sample processing and turnaround time improvements. However, implementation rarely results in total workflow solution with multiple pain points still evident. One of the greatest workflow compatibility issues is related to the SPE elution solvents and LC/MS mobile phases. This incompatibility often results in continued necessity of an evaporation step. While elution solvent volumes are much lower this can still be somewhat of a bottleneck. Here we investigate alternative solid phase extraction chemistries to provide a more complete workflow solution resulting in elimination of the evaporation step prior to LC-MS/MS analysis.

Objectives
This poster will present strategies for automating drugs of abuse extraction, eliminating evaporation steps while maintaining sensitivity using a dedicated low volume sample preparation format.

Methods
A typical drugs of abuse target analyte panel was spiked at various concentrations and extracted from non-hydrolysed and enzymatically hydrolysed human urine. Sample extractions were investigated using polymer-based solid phase extraction: comparing traditional reversed-phase and mixed-mode weak cation exchange chemistries. For increased sensitivity assay miniaturisation was performed using the Biotage® Mikro 2 mg (low volume plate) SPE format. Final low volume extraction protocols were transferred to the Extrahera LV-200 automated sample preparation workstation. UHPLC-MS/MS analysis was performed using a Shimadzu Nexera UHPLC coupled to an 8060 triple quadrupole MS system. Chromatography utilised traditional mobile phases of ammonium formate and formic acid in water and MeOH and separation afforded by a Restek Raptor Biphenyl analytical column.

Results
A drugs of abuse panel including amphetamines, opiates, benzodiazepines, cocaine and other regularly analysed drugs were extracted from urine using polymer-based reversed phase and the corresponding mixed-mode weak cation exchange SPE chemistries in 10 mg 96-well plate format. Strong cation exchange SPE chemistry was ruled out due to necessity of high pH elution conditions which were incompatible with LC mobile phases. Initial evaluations of the polymeric reversed phase chemistry delivered reproducible analyte recoveries, typically greater than 80% depending on drug polarity and wash solvent composition. Further investigation with respect to matrix factors demonstrated very different results. High matrix effects were observed on many mid to non-polar analytes such as the benzodiazepines. Wash solvent, pH and elution solvent optimisation did not adequately remove the matrix effects ultimately ruling out this chemistry option. Weak cation exchange SPE optimisation delivered recoveries great than 80% for all but a few target analytes such as benzoylecgonine and the 7-amino-benzodiazepine metabolites. Matrix factors were substantially better compared to the corresponding reversed-phase chemistry data. Miniaturisation to the Biotage® Mikro 2 mg WCX (low volume plate) format demonstrated excellent scalability. Further optimisation was performed for all steps in terms of pH control, ionic strength and volumes. Final elution volumes of 30 µL were achieved using the optimised method. Comparison of direct injection of the elution solvent to pre-dilution prior to injection for chromatographic peak shapes and injector reproducibility demonstrated the latter approach to be our preferred option. Ultimately, either approach could be used order to allow the elimination of evaporation steps. The extraction protocol was transferred and optimised using the Extrahera™ LV-200 automated sample preparation platform, demonstrating excellent correlation to manual PPM-96 processing. Final method performance and calibration curves demonstrated excellent linearity and coefficients of determination, r2 > 0.99 for all analytes while delivering sub ng/mL LOQs.

Conclusion
The WCX chemistry provided a better approach to drugs of abuse testing while allowing the elimination of evaporation steps for improved workflow. Good correlation was observed scaling chemistry from 10 mg to 2 mg plate options and also between manual and automated processing of the final method.

Tackling the Peptide Isomer Impurity Challenge with High-Resolution Ion Mobility Mass Spectrometry Heidi Vitrac (Presenter) MOBILion Systems

Poster #52b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

INTRODUCTION:
The pace of therapeutic peptide drug development has significantly increased over the last decade. Fueled by technological advancements in structural biology, recombinants and new synthetic and analytical techniques have shortened the time to market and increased the complexity of the peptide therapeutic. Peptides, while demonstrating several advantages over small molecules and larger biologics, including low immunogenicity, high specificity, and low cost, do have drawbacks, most notably stability and half-life. To improve peptide therapeutic biological function and pharmacokinetic profiles, several strategies have been employed, including, amino acid substitution, incorporation of unnatural amino acids, amino acid modifications, and cyclization. However, through the synthesis process, impurities can result that are often difficult to remove by purification alone. Therefore, robust analytical methods for analyzing peptide impurities are required. Hyphenated approaches such as high-resolution ion mobility mass spectrometry (HRIM-MS) have recently emerged as valuable tools for peptide therapeutic impurity analysis. HRIM is an analytical separation performed in the gas phase where ions are drawn through an inert background gas by an electric field. The mobility for these ions is dependent on their charge and shape and is measured as arrival time. In this way, challenging separations, like the separation of peptide isobars and isomers, have been demonstrated.

OBJECTIVES:
The main goal of this work was to leverage a combination of LC, High-Resolution Ion Mobility (HRIM), and QTOF approach to tackle the existing separation challenges of peptide isobaric and isomeric mixtures.

METHODS:
We used the MOBILion HRIM system based on Structures for Lossless Ion Manipulation (SLIM) to assess several types of isobaric and isomeric peptide modifications. Data acquisition was carried out on a high-resolution ion mobility (HRIM) MOBIE™ instrument (MOBILion Systems) coupled to a 6545XT QTOF (Agilent Technologies) with a 1290 Infinity II Autosampler (Agilent Technologies), using a combination of flow injection analysis (FIA) and reversed-phase liquid chromatography (RPLC) separation of standard peptides. For RPLC analyses of deamidated peptides, an Agilent Zorbax Eclipse C18, 50 x 2.1mm, 1.8 µm column at a flow rate of 0.4 mL/min was employed. All FIA and RPLC analyses were conducted using 0.1% formic acid in water and acetonitrile as mobile phases A and B, respectively, and the Agilent Dual Spray Jet Stream ESI Source operated in positive ion mode. The SLIM chamber was maintained at 2.5 Torr throughout the experiments. The SLIM-based HRIM Device was tuned and calibrated to optimize the transmission of ions between the mass range of m/z 300 and 3200. A series of experiments were carried out to determine the optimum traveling wave parameters required to trap and release ions to obtain the maximum resolution and sensitivity. Accurate mass, isotope spacing, ion mobility arrival time distribution, and Collision Cross Section (CCS determination were used for the identification of modified/unmodified peptide mixtures. Data processing, analysis, relative quantification, and visualization were achieved using HRIM Data Processor V1.10.29.1 (HRIM-DP), PNNL Preprocessor Version 4.0 (2022.02.17), Agilent IM-MS Browser.

RESULTS:
Herein we report our use of the MOBIE® HRIM from MOBILion with an Agilent LC-QTOF system to improve therapeutic peptide impurity analysis and demonstrate its applicability for resolving isobaric and isomeric peptide mixtures based on the gas phase separation of D- and L-amino acid isomers and deamidation and isomerization isomers. The novel HRIM method was first qualified using a range of peptide standards. We then evaluated the utility of the LC-HRIM-MS method for quantifying peptide impurities and compared LC-based quantification to quantification in the ion mobility domain. We demonstrate that implementing HRIM into the peptide analysis workflow enables confident separation, detection, and relative quantification of difficult-to-analyze isobaric and isomeric peptides reproducibly thereby eliminating the reliance on MS/MS fragmentation pattern. MOBIE® solves the 0.984 Da mass shift problem by providing an orthogonal separation, reducing the dependency on RPLC separation while maintaining confident identification and quantification of peptide modifications within isobaric/isomeric peptides.

CONCLUSION:
Mass spectrometry has proven extremely useful for analyzing amino acid modifications, facilitating mass determination, and sequencing of peptides. However, numerous challenges persist, including analyzing isobaric and isomeric peptide modifications, where existing separation technology has been limited in its utility. The LC-HRIM-QTOF system used is a powerful combination that can be implemented to enhance existing LC workflows for deeper sample characterization, reduce LC reliance to boost throughput, and add CCS values to existing compound identification.

Development of a Dried Blood Spot Lead Test by ICP-MS to Increase Screening Compliance in at Risk Populations Cody Orahoske (Presenter) University of California San Francisco

>> POSTER (PDF)

Poster #59a View Map

This poster will be attended on Thursday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:
Despite significant progress decreasing blood lead levels among US children 1-5 years of age, demonstrated by a 97.5th percentile of 3.5 µg/dl, lead poisoning remains a public health threat. Lead exposure disproportionately affects Black children, and those below poverty levels, in Medicaid, and living in housing units built before 1978. To improve public safety and testing compliance in children younger than 2 years of age, sample collection can be done by using filter paper and dried blood spots. These cards offer the advantage of easy handling and a less invasive collection method. Lead testing from the dried blood spot is comparable to the gold standard detection of lead from a venous whole blood samples although may be prone to contamination and other interferences. We describe a robust and reproducible test which can be used to measure lead from dried blood spots.

Objective:
The primary objective of this study was to develop a method using dried blood spot cards for lead testing using inductively coupled plasma-mass spectrometry (ICP-MS).

Methods:
This method was developed on a Thermo Fisher iCAP RQ and TQ ICP-MS (Thermo Fisher Scientific, Waltham, MA, USA) in kinetic energy discrimination (KED) mode. The method was calibrated with certified reference material traceable to NIST SRM 3128 (VHG labs, Manchester, NH, USA) in whole blood spotted in Whatman 903 cards at various concentrations and dried for at least two hours. Briefly, 5 ml of extraction buffer was added to a 6mm filter paper disc, followed by a 1-minute vortex at 2,000 RPM, incubation at room temperature for 30 minutes and centrifugation at 4,000 RPM for 5 minutes. Samples were loaded onto an Elemental Scientific Inc. (ESI, Omaha, NE, USA) SC-FSAT sample introduction system and subjected to analysis by the ICP-MS. To assess the performance of the method, the following characteristics were established: linearity, reproducibility, and accuracy. Accuracy and linearity across the measurement range were assessed using a reference standard solution traceable to NIST CRM 3128 (Inorganic Ventures, Christiansburg, VA, USA). Reproducibility and accuracy were assessed using used UTAK controls (Valencia, CA) and 16 patient samples with lead values obtained in a whole blood method.

Results:
The assay is linear across the analytical measurement range of 1 to 100 µg/dl, and recovery of the linearity materials ranged from 92% to 102%, with an average SD of ± 0.8 µg/dl. In 16 patient samples with concentrations ranging from 1.4 to 19.5 µg/dl run in six replicates, the coefficient of variation (CV) range was 0.6% to 21.1%. In these samples, the bias ranged from -4.4 µg/dl to 0.1 µg/dl. In controls recovery ranged from 88% to 100% and CV ranged from 2.6% to 6.0%. Finally, this method successfully quantified five proficiency testing samples from the Dried Blood Spot Lead Proficiency Testing Program (Wisconsin State Laboratory of Hygiene, University of Wisconsin, Madison, WI).

Conclusion:
The dried blood spot method was reproducible and comparable to the routine testing method in whole blood. This dried blood spot lead test meets the performance criteria to be validated as a clinical test in pediatric patients.

Determination of Free Thyroxine from Human Serum Using BioSPME Sample Preparation Prior to LC-MS/MS Analysis Olga Shimelis (Presenter) MIlliporeSigma

Poster #60a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

Introduction:

Absolute free thyroxine concentrations remain relatively constant, approximately 0.02% of total thyroxine (tT4) is available in circulation in the free form. The vast majority is bound to thyroxine-binding globulin and to a lesser extent albumin and transthyretin. Direct immunoassays, the most common test performed at clinical laboratories, suffer from interferences and lack of specificity. In a 2002 study of > 5,000 patients, reported this incident occurring in ~0.5% of samples (Ismail AAA et al. 2002, Ann Clin BioChem). Liquid chromatography tandem mass spectrometry (LC/MS/MS) methods have been developed over the past 10 years to afford improved specificity and accuracy. The current standard for separation of free hormones prior to analysis has been equilibrium dialysis (ED). In general, ED is a lengthy process, on average overnight at minimum. A novel sample preparation has been utilized to alleviate this. Solid phase microextraction, SPME, has been developed into a 96-pin device termed BioSPME to prepare samples in under one-hour.

Objective:

A collaboration was established between MilliporeSigma and ARUP Laboratories to investigate the results of sample preparation by a BioSPME device or ED prior to LC/MS/MS analysis for measurement of free thyroxine in serum.

Methods:

The free thyroxine (fT4) and/or free triiodothyronine (fT3) were analyzed on Agilent 1290 LC utilizing an Ascentis Express Biphenyl (10 cm x 2.1 mm, 2.7 µm) connected to an AB Sciex 6500 QQQ. Quantifier and qualifier transitions were utilized for unlabelled and isotopically 13C6-labelled thyroxine (T4), triiodothyronine (T3), and reverse triiodothyronine (rT3). The extracted calibration standards were used to directly report the concentrations of fT4 and/or fT3.
Initial method development utilized bulk serum that was tested by an external clinical laboratory for independent determination of fT4 and fT3 by a validated ED-LC-MS/MS method. These samples were additionally used to perform reproducibility assays of the method.

The BioSPME sample preparation method consisted of using 200 µL volume samples and a C18 coated 96-pin device with a Hamilton Starlet system for automation. The method included multiple steps of transferring the device between four well plates (condition, wash, sample, and desorption). The sample plate consisted of calibration standards prepared in 7.5 mM HEPES at pH 7.5 and serum samples which were diluted with 5% (v/v) of 1.15 M HEPES to adjust the pH. Extracted samples were desorbed into 40 µL of methanol containing internal standards. The samples were then diluted with 40 µL of water by the autosampler prior to injection.

Reinjection of extracted samples was investigated using 29 serum samples and 7 calibrators.

Serum samples for robustness testing were provided in conjunction with ARUP Laboratories (Salt Lake City, UT) and were previously determined by equilibrium dialysis-LC-MS/MS using the method described by Bingfang Yue (Yue, B, Rockwood, A, et al. 2008, Clinical Chem).

Results:

Six commercial serums were tested on five (n=4 per extraction) separate occasions to test reproducibility of the method for fT4 and fT3. On average, the interday %CV for fT4/fT3 were as follows: 10.9/9.2%, 5.5/9.2%, 8.6/6.9%, 10.2/9.5,% 4.0/8.5%, and 7.8/7.2%.

A series of different sets of serum samples were investigated. The first set of 24 (n=3), a correlation of ED-LC-MS/MS to BioSPME-LC-MS/MS yielded a relationship of y = 0.738x + 0.322, R2 = 0.867. The average % CV for the fT4 concentration was 8%. On average, fT4 values for BioSPME-LC-MS/MS were 3% lower than ED-LC-MS/MS.

In a second set of 45 samples (n=1), the overall correlation was y = 0.781x + 0.464, R2 = 0.819. On average, the fT4 values using BioSPME-LC-MS/MS were 22% lower than ED-LC-MS/MS. Additional method development is on-going to increase the accuracy of the BioSPME-LC-MS/MS method.

A study involving 29 serum samples and 7 calibrators were reinjected non-sequentially twice. On average, there was a 4.1% difference in concentration.

The LLOQ of the extracted calibrators was 1 pg/mL with a 40 µL injection with an % CV of 10.9% and 19.8% (quantifier and qualifier). The peak integration ratio for the LLOQ for the quantifier/qualifier was 1.00, % CV 14.6%. The sensitivity was achieved by replacing the 20 µL standard injection loop on Agilent 1290 LC instrument with a 100 µL loop to allow for larger injection volumes.

Conclusion:

A BioSPME extraction method prior to analysis by LC-MS/MS was developed, and the evaluation results showed strong correlation against equilibrium dialysis for determination of free thyroxine (fT4) from serum samples. The BioSPME method was automated by using a Hamilton robotic system and can be adapted to other robotic liquid handlers that have gripper functionality. The time to process one 96-well plate was less than an hour. The developed LC-MS/MS detection method included a built-in preconcentrated sample (5x) without dry-down steps and the ability of get repeat injections with consistent results.

Global Metabolomics and Lipidomics Platform for Ultra-High Performance Molecular Phenotype Analysis Shuang Zhao (Presenter) The Metabolomics Innovation Centre

Poster #68a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour in the Exhibit Hall.

Introduction
Comprehensive and quantitative characterization of molecular phenotype of biological samples is crucial for biological studies and health research. Dealing with detection and quantification of small molecules, metabolomics and lipidomics become emerging tools to study molecular phenotype in a time-sensitive and treatment-sensitive manner. Due to the significant differences in chemical and physical properties of metabolites and lipids, these two classes of compounds are analyzed separately. The accurate, reliable and meaningful molecular phenotype characterization relies on using proper and high performance metabolomics and lipidomics approach. Here we present a deep and global metabolomics and lipidomics platform for molecular phenotype analysis. The proposed technology was applied to different types of samples in proof-of-concept studies.

Methods
The metabolomics was performed using 4-channel chemical isotope labeling (CIL) LC-MS approach. Briefly, after metabolite extraction, samples were aliquots to four parts and derivatized with a pair of isotope reagents (i.e., 12C/13C-reagents) for each channel, followed by LC-MS analysis. Data processing was then preformed using a dedicated software IsoMS Pro.

The lipidomics was performed using in-depth global lipidomics approach. A set of rationally designed stable isotopic labeled internal standards (SIL-IS) was mixed with each sample, followed by lipid extraction. LC-MS and LC-MS/MS analyses were performed in positive and negative ion mode for each sample. All acquired data was processed by software LipidScreener.

The data from metabolomics and lipidomics can be interpreted either separately or combined together to achieve ultra-high coverage.
 
Preliminary Results
In the CIL LC-MS metabolomics, four submetabolomes were analyzed using isotopic reagents, i.e., amine/phenol, carboxyl, hydroxyl, and carbonyl submetabolome. With optimal design of the reagents, concomitant improvement in separation, detection and quantification can be achieved. To enable comprehensive identification, a three-tiered and dual-identification approach was employed: in tier 1 and 2, high confidence results can be generated using authentic standards or pathway-related metabolites, respectively; in tier 3, MS search was applied. With benefits from both labeling and comprehensive identifications, high performance analysis can be realized with much stronger ability for scientific discovery.

In the in-depth lipidomics, unique SIL-IS were created to improve the quantification of common lipid types. MS and MS/MS data were acquired to enhance both lipids coverage and identification. A three-tiered identification approach was developed. In tier 1 and 2, MS/MS data was used for positive identification with different score thresholds; in tier 3, MS data was used for putative match. With high coverage and better quantification using unique SIL-IS, high performance lipidomics can be accomplished.

Workflows of analyzing common samples have been developed. As an example, 18 human serum samples were analyzed using this technology. In metabolomics, 9179 ± 71 metabolites per sample were detected and relatively quantified. Among them, more than 1500 metabolites were identified with high confidence, covering over 100 metabolic pathways. In lipidomics, 8385 ± 266 lipids per sample were detected. Among them, more than 1000 lipids could be identified using RT, MS and MS/MS. Using this technique, a total of more than 2500 metabolites and lipids could be identified with high confidence and relatively quantified with high accuracy and precision, indicating an exceptionally high coverage and performance for molecular phenotype characterization. In the presentation, the analysis power of using integrated dataset of metabolomics and lipidomics for studying samples with different phenotypes will be shown.

Conclusions
A global metabolomics and lipidomics platform was developed for deep characterization of molecular phenotype.

Aerogel-Based Combi-Matrix for Cancer Diagnosis with LDI Mass Spectrometry Tae Gyeong Yun (Presenter) Yonsei University

Poster #68b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

Introduction:
Matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) has been widely used in clinical fields for the analysis of large biomolecules. However, MALDI-ToF MS on small molecules are limited due to the fragmented matrix molecules during ionization process. Furthermore, the analyte crystals usually form inhomogeneous shape, so the mass signals vary by the sample spot morphology even in the constant laser irradiations. Therefore, the quantitative analysis has been limited for conventional MALDI-ToF MS. To overcome this limitation, various solid matrices were suggested to replace the organic matrices by using their structural stability in laser radiation. Moreover, the concept of combi-matrix also reported by mixing organic matrices and other type of materials to enhance the mass spectrometry results.

Objectives:
In this study, a combi-matrix system was prepared by mixing organic matrix with inorganic silica aerogel. The nanoporous structure and the unique thermal insulating properties of silica aerogel was demonstrated through the material characteristic analysis. Furthermore, Laser desorption/ionization mass spectrometry (LDI-MS) based on combi-matrix was applied to the quantitative analysis of dodecanoyl-L-carnitine (DC) using patient samples for clinical diagnosis of colon cancer.

Methods:
Silica aerogels for combi-matrix component was synthesized from sodium silicate precursor by two-step sol-gel process with ambient pressure drying. The combi-matrix was prepared by mixing silica aerogel and organic matrix (α-4-hydroxycinnamic acid, CHCA) in ethanol condition. The optimized ratio was confirmed by calculating S/N ratio from LDI-MS results of model analytes. The feasibility of combi-matrix for quantitative analysis was confirmed by fluorescence image test, shot-to-shot, and spot-to-spot reproducibility test. The difference in material perspective between organic matrix and combi-matrix was confirmed by thermal analysis using DSC. The quantitative analysis on small molecules with LDI-MS using combi-matrix conducted in various sample condition including patient real samples.

Results:
Synthesized silica aerogel was prepared into powder form. Average hydrodynamic radius of silica aerogel was measured to be 100-200 nm by dynamic light scattering (DLS) analysis. A typical pore structure of aerogel was confirmed by the scanning electron microscope (SEM) and the Brunauer, Emmett, and Teller (BET) method. The morphological data of aerogel was estimated with an average pore size of 9.3 nm and a porosity greater than 82.1%.

The applicability of silica aerogel for LDI-MS combi-matrix was demonstrated step-by-step by mixing it with a CHCA organic matrix. Silica aerogel itself resulted no mass peaks from model amino acid (histidine). However, the silica aerogel could be used for the elimination of mass peaks of the organic matrix by the mixed zone of matrix and analyte within the nanopores of aerogel. The component ratio between organic matrix and silica aerogel was controlled in the range from 1:0.2 to 1:2.6×10^-6 (weight per weight, w/w). The optimal ratio was estimated to be 1:1.6×10^-3 with the highest signal-to-ratio of 154.2 (a.u.).

The homogeneous and dense distribution of analytes for quantitative analysis was demonstrated by the fluorescence imaging of sample spot. The morphology of crystals in sample spot was compared at different concentrations of silica aerogel. The coffee-ring effect on fluorescence intensity was minimized as the aerogel component was increased to the former optimal ratio of combi-matrix. Additionally, the shot-to-shot reproducibility (96.0%) and spot-to-spot reproducibility (90.4%) were confirmed through the combi-matrix.

Investigation on the thermal properties of silica aerogel in combi-matrix was conducted using differential scanning calorimetry (DSC). The DSC analysis showed that the low thermal conductivity of silica aerogel reduces the thermal dissipation of laser energy through the sample plate. In addition, using the Gibbs-Thomson equation for organic matrix (CHCA), the average crystal radius of CHCA in the combi-matrix system was calculated to be smaller than that of the bulk state. This phenomenon represents the silica aerogel induces a nanoscale crystal of the organic matrix.

LDI-MS analysis using combi-matrix was conducted for the quantitative analysis of a biomarker for colon cancer, dodecanoyl-L-carnitine (DC). A standard curve obtained a linear correlation in the wide concentration range with a linearity factor over 0.94 for buffer and spiked serum conditions, respectively. A clinical diagnosis of colon cancer was demonstrated using a combi-matrix with real samples from healthy volunteers and cancer patients. The results from real samples were compared with the conventionally used LC-MS. The correlation of two experimental results was statistically analyzed using Bland-Altman plot and Passing-Bablok regression. Two methods distributed within the 95% confidence level, and the Spearman correlation coefficient was 0.947 (p<0.0001). Therefore, the LDI-MS with combi-matrix and conventional LC-MS results in statistically coincidence.

Conclusion:
In this work, a combi-matrix was presented based on the mixture of organic matrix and nanoporous silica aerogel as an inorganic component for quantitative analysis using LDI-MS instrument. The optimal ratio for combi-matrix was determined and small molecule analysis was conducted. The feasibility of quantitative analysis was confirmed by various ways, including fluorescence distribution, reproducibility test, and thermal property analysis. The combi-matrix was applied for the quantitative LDI-MS analysis of a biomarker for colon cancer, dodecanoyl-L-carnitine. Furthermore, by using serum samples from healthy volunteers and patients, clinical diagnosis of colon cancer was demonstrated using combi-matrix system.

The MAW Method: An Efficient Monophasic Extraction Method for Bacterial Lipidomics Kingsley Bimpeh (Presenter) University of Georgia

Poster #69b View Map

This poster will be attended on Thursday at 12:30 for 1 hour in the Exhibit Hall.

Introduction:
Usually overlooked, a good lipid extraction method is essential for any lipid analysis. Different extraction methods are available, but each enriches different types of lipids due to their diversity in structure and polarity. Generally, these methods rely on a methanol/chloroform/water solvent system that causes phase separation and were developed with mammalian lipids from serum, plasma, or tissue in mind. While they are suitable for finding the major lipids in bacteria, there are downsides in utilizing these methods such as poor reproducibility, inability to support high-throughput experiments, and the use of a widely known carcinogen, chloroform.

Objectives:
The main goal of this work was to develop a chloroform-free single-phase lipid extraction method based on methanol, acetonitrile, and water (MAW method) for the recovery of lipids from bacteria that circumvents the issues affecting the reproducibility and throughput of biphasic liquid-liquid extractions.

Methods:
Different combinations of acetonitrile/methanol ratios ranging from 1:4 to 4:1 were used to extract the major species of endogenous lipids (i.e., PGs, DGDGs, and LysylPGs) from the S. aureus model system. All analyses were performed with an online HILIC column and a Waters Synapt XS traveling-wave ion mobility-mass spectrometer (TWIM-MS). Subsequent mass spectrometry data were submitted to Progenesis QI (Waters) for alignment, peak picking, and multivariate statistical analysis. Given the amphipathic nature of polar phospholipids, the impact of acidification on extraction yield was examined. The optimized conditions for the single-phase extraction, consisting of 1:4 ACN/MeOH with 0.5% (v/v) acetic acid, were tested against the biphasic Bligh & Dyer (B&D) extraction method for the recovery of LysylPGs, DGDGs, and PGs in S. aureus strain JE2-Dap2. For further analytical quantitation, we determined the absolute lipid recovery using nonendogenous internal standard lipids, the limit of detection (LOD), and quantitation (LOQ) following the MAW and B&D extractions. We evaluated whether the MAW method could be scaled down into volumes compatible with 96 well plates and, to a greater extent, simplified by eliminating the drying and reconstitution steps prior to LC-MS analysis. To demonstrate the suitability of the high-throughput implementation of the MAW method for profiling, we performed untargeted lipidomics on daptomycin-resistant and susceptible strains that were extracted in 96-well microplates using the MAW method.

Results
The optimum solvent conditions for identifying all the essential lipids in S. aureus were 1:4 (%v/v) acetonitrile/methanol. Acidification enhanced the yield of LysylPGs and DGDGs, confirming our initial hypothesis that acidification of anionic and cationic phosphatides disrupts the ionic interaction present between these lipids and the ionized surrounding environment. Comparison of the MAW method against the Bligh & Dyer extraction showed minimal differences in the extracted amounts of LysylPGs, DGDGs, and PGs, but a 3-fold improvement in reproducibility. The extraction recovery of isotope-labeled internal standards confirmed that the MAW method provided comparable recoveries (ca. 90%) to the Bligh & Dyer method. Calibration curves generated for the limit of detection and the limit of quantification of PGs further reveal that the MAW method is sensitive and requires less bacteria for lipid analysis. Furthermore, the lipid peak areas of the small-scale extraction approach were statistically indifferent and strongly correlated with those of the 3.5 mL large-scale extraction when using a total volume of 175 µL (R2 > 0.99; Pearson P < 0.0001). Further simplification by eliminating the drying and reconstitution steps before LC-MS analysis of lipid extracts resulted in higher-intensity signals. Ultimately, the results from the untargeted lipidomics of the daptomycin-resistant and susceptible strains were the same as those of previous lipidomic measurements.

Conclusion:
This work presents a single-phase extraction method, the MAW method, for the recovery of lipids in bacteria. The MAW method demonstrates significant advantages, including ease of use, increased sensitivity coupled with greater reproducibility when compared to the Bligh & Dyer method.

Urinary 2-Cyanoethylmercapturic Acid for Smoking Status Classification Deepak Bhandari (Presenter) Centers for Disease Control and Prevention

Poster #72b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

INTRODUCTION: Cotinine is a widely used biomarker for classifying cigarette smoking status; however, cotinine cannot differentiate use of combustible vs noncombustible tobacco products. Herein, we evaluated urinary 2-cyanoethylmercapturic acid (2CyEMA), a metabolite of acrylonitrile, as a complementary urinary biomarker for assessing smoke exposure.

OBJECTIVES: The primary objective of this study is to establish a reference cutoff value of urinary 2CyEMA for differentiating users of cigarettes from users of noncombustible tobacco product.
METHOD: We performed a receiver operating characteristic (ROC) curve analysis of a representative sampling of the adult U.S. population (NHANES 2011-2016, the special smoker subset, ages >20) for identifying optimal cutoff concentration by maximizing Youden’s J index. Smoking status was categorized based on the recent tobacco use questionnaire.

RESULTS: The cutoff concentration was 7.32 ng/ml with high sensitivity and specificity (≥0.925). When stratified by demo¬graphic variables, the cutoff concentrations varied among subgroups based on age, sex, and race/Hispanic origin. Non-Hispanic Blacks had the highest cutoff concentration (15.3 ng/ml), and Hispanics had the lowest (4.63 ng/ml). Females had higher cutoff concentrations (8.80 ng/ml) com¬pared to males (6.10 ng/ml). Among different age groups, the cutoff concentrations varied between 4.63 ng/ml (21–39 years old) and 10.6 ng/ml (for ≥60 years old). We also explored the creatinine adjusted cutoff values.

CONCLUSIONS: Urinary 2CyEMA is a selective smoke exposure biomarker that effectively distinguishes between people who use cigarettes from those using noncombustible tobacco products, including ecigarettes. Measuring urinary 2CyEMA in conjunction with cotinine can unequivocally assess tobacco smoke exposure.

Disclaimer: The views and opinions expressed in this report are those of the authors and do not necessarily represent the views, official policy or position of the U.S. Department of Health and Human Services or any of its affiliated institutions or agencies. The use of trade names is for identification purposes and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, or the U.S. Department of Health and Human Services.

4D Proteomic Profiling of 200 Individuals Plasma with DIA-MS and Internal Standards for a Health Surveillance Panel-A High-Throughput Workflow Qin Fu (Presenter) Cedars Sinai Medical Center

Poster #76b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour in the Exhibit Hall.

Introduction
There is a profound need in establishing a defined plasma proteomic baseline of a healthy population and establish normal reference ranges for quantifiable peptides. Human plasma contains thousands of proteins and makes contact with virtually all cells in the human body. Therefore, the plasma proteome is reflective of an individual’s health status. It is feasible to develop an analytic method for a Health Surveillance Panel (HSP) comprised of selected and relevant plasma proteins. We have selected plasma proteins with important biological functions and involvement in diverse pathways with 50% being FDA or LDT tests. We investigated 4D proteomic profiles of 200 healthy individuals by employing the following: 1) automated sample preparation; 2) 83 spiked in stable isotopic labeled heavy peptides (representing HSP proteins) as internal standards; 3) timsTOF Pro dia-PASEF (Parallel Accumulation Serial Fragmentation); 4) dia-PASEF MS acquisition and implementation of automated LC-MS/MS quality controls; 5) novel library building algorithm to calibrate and normalize CCS (collisional cross-sectional) values; 6) CCS-enabled DIA-NN for neural networks search algorithm (Tims DIANN) for analyzing data.

Objectives
This is the first in-depth study of 4D plasma profiling in a healthy population. Development of a robust, precise, quantitative and automated 4D proteomic profiling workflow to profile healthy individuals with dia-PASEF MS methodology, spiked internal standards and to establish the proteomics healthy population baseline (n=200).

Methods
Development of the workflow, reproducibility and linearity of the tryptic peptides were carried out using a gender-pooled plasma from 100 females and 100 males. Protein denaturation, reduction, alkylation, digestion and desalting were performed on a Beckman i7 automated workstation. Digested individual plasma samples (800 ng) with 83 SIL heavy peptides (57 HSP proteins) were injected onto an PepSep column attached to an EVOSEP One coupled to a Bruker timsTOF Pro mass spectrometer. Plasma samples were injected in triplicate. The workflow was validated on plasma samples from 200 healthy individuals enrolled in Coronavirus Risk Associations and Longitudinal Evaluation (CORALE) Study and run robustness was evaluated based on quality control runs of predigested plasma (n=5) as an end-to-end workflow control (system suitability).

Results
Healthy individuals’ plasma (n=200) were baseline of the CORALE Study and who self-reported as being healthy and were not COVID-19 positive comprising 45% female, age ranged 21 to 69 years and composed of four ethnic groups: 7% African American or black, 22% White Hispanic, 37% Asian and 34% Caucasian. For data analysis using our newly developed DIA data analysis suite that builds spectral libraries from either real-time data or existing peptide libraries from different sources (e.g. multiple in-house DDA libraries, and libraries in the public domain). DIA data was analyzed using our improved CCS-enabled version of DIA-NN that calculates ion mobility-based scores for both target and decoy precursors and build features for deep learning. The spectral library tool calibrates CCS values and re-normalizes fragment ion intensities, which is used in the CCS-enabled DIA-NN for neural networks. We applied our complete workflow to plasma from 200 individuals. The pooled QC plasma (system suitability) was used for MS and full workflow performance. The reproducibility, CV, LLOQ and linearity were established for 83 SIL peptides in the plasma matrix. The 4-D proteomic baseline profiled 200 baseline healthy individuals using timsTOF dia-PASEF MS methodology, novel data analysis and bioinformatic package including library building/calibrating and an improved CCS-enabled DIA-NN search algorithm.

Conclusion
Development of a robust, precise, quantitative, and automated proteomic profiling workflow with HSP SIL peptides as internal standards using a dia-PASEF -MS methodology, novel library building and CCS-enabled DIA-NN algorithm to profile 200 individuals 4D proteomic plasma baseline.