I hold a bachelor degree in Pharmacy from the Hebrew University of Jerusalem, Israel. I did my PhD at the same university specializing in nanocscience and drug delivery with Prof. Shlomo Magdassi (Chemistry Department). During my postdoctoral training with Prof. Richard Zare at Stanford University, USA I worked on ambient mass spectrometry imaging of clinical specimens, mostly employing Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI).
I started a tenure track position as a Senior Lecturer (Assistant Professor) at the School of Pharmacy, the Faculty of Medicine, the Hebrew University of Jerusalem, Israel, in September 2019.

I studied biology at the University of Zurich, Switzerland, and made my PhD in Molecular Plant Physiology in the lab of Prof. Enrico Martinoia, focusing on ABC transporters and comparative molecular phylogenetics. Subsequently, I became as researcher at the University Hospital of Zurich, where I was involved in establishing an LC/MS-based assay for the peptide hormone hepcidin and in projects studying Fabry Disease mechanisms. Biological processes, bioanalysis, clinical applications and the use of informatics to improve workflows have been my constant research interests. With this background I am now working as a senior researcher at the Singapore Lipidomics Incubator (SLING), heading our new data team that is focusing on developing workflows and software pipelines for the analytical data processing, QA/AC and exploration of the diverse lipidomics datasets generated by our lab.

Irene van den Broek has a PhD in analytical chemistry from the Utrecht University, the Netherlands and over 10 year experience in quantitative mass spectrometry for clinical and proteomics applications.

Distinguished Professor I. Fournier (IF, PRISM, U1192, Lille) is senior member of the University Institute (IUF) with a chair in Clinical Mass Spectrometry. She is currently co-director of PRISM lab INSERM U1192. Prof. Fournier is a specialist of Mass Spectrometry applied to clinics and proteomics. She was pioneer a novel technology in Europe of molecular imaging namely MALDI Mass Spectrometry Imaging in 2002 (Fournier et al., Neuroend. Letters 2003) and in 2007 demonstrated that MALDI MS Imaging could be perform from archived Formalin fixed and Paraffin Embedded hospital tissue samples (Lemaire et al., J. Proteome Res 2007). In 2005, she developed the multiplex targeted imaging, Tag-mass technology (Lemaire J. Proteome Res. 2007; Gagnon et al., Prog Histochem Cytochem 2012). In 2013, she introduced the concept of the spatially-resolved tissue proteomic and applied this technology on different cancers (Longuespee et al., Cancer Metastasis Rev 2012; Quanico et al Chem Com, 2015, Simeone et al., Semin Cancer Biol., 2018). Since 2011, Pr. I. Fournier with Pr. X. Roucou have evidenced the Ghost proteome (Samandi et al., Elife 2017, Delcourt et al., EBioMedicine et al., 2017, Brunet et al., Nucleic. Acid. Res., 2019). In 2014, she performed the development of a novel technology based on mass spectrometry and allowing to realize in vivo real time tissue molecular imaging and for guiding surgery, with an instrument named SpiderMass (Saudemont et al., Cancer cells, 2018; Ogrinc et al., Nature Protocols, 2019).

David C. Muddiman is the Jacob and Betty Belin Distinguished Professor of Chemistry and Director, Molecular Education, Technology, and Research Innovation Center (METRIC) at North Carolina State University in Raleigh, NC. Prior to moving his research group to North Carolina State University in 2006, David was a Professor of Biochemistry and Molecular Biology and Founder and Director of the Proteomics Research Center at the Mayo Clinic College of Medicine in Rochester, MN. Prior to this appointment, David was an Associate Professor of Chemistry at Virginia Commonwealth University. It was there that he began his professional career as an assistant professor with an adjunct appointment in the Department of Biochemistry and Molecular Biophysics and as a member of the Massey Cancer Center in 1997. These academic appointments followed a postdoctoral fellowship at Pacific Northwest National Lab

In 2018, Prof. David Muddiman was named the Director of the Molecular Education, Technology, and Research Innovation Center. METRIC provides NC State researchers and University partners with world-class state-of-the-art measurement science facilities across four buildings throughout campus, encompassing three key molecular characterization technologies including mass spectrometry, magnetic resonance (NMR and EPR), and X-Ray Crystallography (small molecule and macromolecular).

The Muddiman Group focuses on the development of innovative mass spectrometry measurements to solve important biological problems. Research projects include developing an ambient ionization method called IR-MALDESI and its application to a diverse range of materials, including soft tissue, plants, textiles and bone. Moreover, the group is working on advanced separations to enable detailed characterization of complex biological samples.

Wojciech Michno has a highly international background. He is a United World College (Norway) scholar and a Macalester College (USA) alumnus. At the university he completed two degrees in parallel, one in Chemistry and one in Neuroscience, with a focus on molecular neurobiology. Wojciech did his master’s thesis in pharmacology, and recently defended his PhD in analytical neurochemistry at Sahlgrenska Academy, University of Gothenburg, Sweden. The focus of his work is Alzheimer’s disease.

Prof Roessner has obtained her Diploma in Biochemistry at the University of Potsdam and the John Innes Institute in Norwich, UK after which she pursued a PhD in Plant Biochemistry at the Max-Planck-Institute for Molecular Plant Physiology in Germany, where she developed novel GC-MS methods to analyse metabolites in plants. Together with the application of sophisticated data mining, the field of metabolomics was born and is today an important tool in biological sciences, systems biology and biomarker discovery. In 2003 she moved to Australia where she established a GC-MS and LC-MS based metabolomics platform as part of the Australian Centre for Plant Functional Genomics for which she led the node at the University of Melbourne. In addition, since 2007, Prof Roessner has been involved in the setup and lead of Metabolomics Australia (MA), a federal and state government funded national metabolomics service facility and now leads the MA node at the University of Melbourne. In 2013, Prof Roessner was awarded an Australian Research Council Future Fellowship to establish her own research program applying Imaging Mass Spectrometry for spatial metabolite and lipid analyses to understand root metabolism under salinity stress. Currently her research program uses metabolomics and lipidomics technologies to decipher how plant roots interact with beneficial microbes under abiotic stress conditions. In 2018, Prof Roessner took up the position as Head of School, School of BioSciences, University of Melbourne.

Join us for our next FeMS Happy Hour! We will be following our usual format with small group breakout room discussions followed by Ute Roesnner presenting on Resilience, and a time for Q&A and Discussion. All are welcome!

Dr. Carmen Wiley was the 2019 - 2020 President of AACC. Recently she returned to the clinical practice of laboratory medicine and serves as Clinical Medical Director at Incyte Diagnostics. In this role, she supports the new clinical lab facility located in Spokane Valley and acts as the Medical Director over the clinical labs at Providence Sacred Heart Medical Center, Holy Family, My Carmel, and St. Joseph’s.

Dr. Wiley holds a Bachelor’s degree in Chemistry from the University of Minnesota, a Master’s degree in Organic Chemistry from the University of Washington, a Doctoral degree in Organic Chemistry from the University of Washington, and was a COMACC Accredited Fellow at the Mayo School of Medicine. She is board certified by American Board of Clinical Chemistry (ABCC) and a Fellow of the Academy of the American Association of Clinical Chemistry (FAACC).

Most recently, she was the Chief Medical Officer of a start-up company based in Oakdale, MN. Previously, she was a Regional Manager of Scientific Affairs – Cardiac at Roche Diagnostics. In this role, she was responsible for leading and developing the Medical & Scientific Liaisons in their relationships with the medical/scientific community, with the objective of critical scientific exchange including medical/scientific education. Dr. Wiley played a key role in providing support to healthcare professionals as well as internal Roche scientific groups and local business teams. Dr. Wiley was the Scientific Director at PAML where she was responsible for the medical and scientific oversight of all laboratory testing and oversaw all aspects of PAML’s research and development program. Prior to that, Dr. Wiley was Co-Director of Chemistry, Immunology, and Point of Care at Providence Health and Services, Sacred Heart Hospital in Spokane, WA and the Head of Clinical Chemistry in the Division of Laboratory Medicine and Pathology at the Marshfield Clinic in Marshfield, WI.

Dr. Wiley, her husband of 21 years and two kids enjoy time spent with their three dogs. She loves hiking, camping, and knitting. She is a native to Minnesota, but now considers Spokane, WA her home.

Dr. Bystrom serves as Translational Laboratory director at Cedars-Sinai, where he is responsible for novel biomarker validation and commercialization. Prior to joining Cedars-Sinai, he directed research and development at Cleveland Heart Lab and commercialized a multiplex proteomic diagnostic test focusing on HDL functionality.

What does clinical chemistry have to do with industry? What opportunities are available for me as a clinical chemistry? Join in our live interview of two clinical chemists who will share their career journey towards earning their clinical chemistry certification and their current position in various industries. Bring your questions and be ready for the networking opportunity after the interview!

I am currently a clinical chemistry fellow at Johns Hopkins University School of Medicine. My clinical chemistry fellowship started at Vanderbilt University Medical Center before transitioning to JH. I received a B.S degree in Clinical Laboratory Sciences from the University of Nevada, Las Vegas where I began research in laboratory medicine and toxicology. Before returning to school to earn my PhD, I served as a medical laboratory scientist at Harborview Medical Center, University of Washington in Seattle, WA in the area of hematology and coagulation. My PhD is in Pharmacology and Toxicology from Michigan State University, where my research was on the adipose tissue's adrenergic control of vasoconstriction. I completed a research postdoctoral fellowship at Yale University in Laboratory Medicine, where I studied RNA-methylation mechanisms of hematopoietic malignancies and completed collaborative research projects in clinical chemistry at the Yale New Haven Hospital on procalcitonin utility and establishing reference intervals.

Steven E. Conklin Lopez received his B.S. in Chemistry (2013) from the University of Puerto Rico – Río Piedras Campus and completed his PhD training (2019) at Duke University under the mentorship of Prof. Katherine J. Franz. As a graduate student, his research focused on understanding the role of metals and metal coordination in the chemical and biological activity of antimicrobial peptides. Currently, he is a Clinical Chemistry Fellow in the Pathology Department of the Johns Hopkins University School of Medicine.

I am a second-year fellow in the Clinical Chemistry Fellowship at Mayo Clinic. Before moving to Minnesota for fellowship, I completed my doctoral degree in Clinical-Bioanalytical Chemistry at Cleveland State University. CSU provided me with many great opportunities to learn about clinical chemistry and to be involved in the clinical chemistry community. I look forward to continuing to be an active contributor to the field in the future.

My name is Nicholas Larkey and I am currently a 2nd-year Clinical Chemistry Fellow at Mayo Clinic.

Dr. Benjamin Owusu is a Senior Clinical Chemistry Fellow at the University of Washington School of Medicine. Benjamin received his PhD degree in Biochemistry and Molecular Genetics and went on to do a one-year postdoctoral research fellowship at the University of Alabama at Birmingham (UAB). He obtained his Bachelor of Science degree in Biochemistry at the University of Ghana, where he also served as a Teaching Assistant at the Department of Biochemistry upon completion of his undergraduate training. He later moved to Emory University School of Medicine in Atlanta, GA on a visiting research scholarship prior to starting his doctoral training at UAB. His research over the years span across cancer, cardiovascular, hematological and endocrinological diseases. In addition to clinical service and research, Benjamin is passionate about teaching and mentoring, particularly in STEM education.

Which clinical chemistry program is right for me? What should I expect from the different programs? Join us in our last session of the series where you can ask your questions about the experiences of current and recent Fellows from three different clinical chemistry programs.

Christina R. Ferreira works as Lipidomics Scientist in the Metabolite Profiling Facility at Purdue University. Her main research interest is the application of the MRM-profiling method for the exploratory analysis of lipids and metabolites in developmental biology models. She created of the MRM-profiling method and contributes to diverse projects from the Cooks lab related to further developing this method. Dr. Ferreira also supports the application MRM-profiling in research projects served by the multi-user Purdue Metabolite Profiling Facility at Bindley Bioscience Center. She iss the project manager for a large effort (Purdue Make-It System) for high-throughput screening and analysis of chemical reactions using DESI-MS.

R. Graham Cooks is the Henry Bohn Hass Distinguished Professor in the Department of Chemistry at Purdue University. He has served as major professor to 140 PhD students. Dr. Cooks’ was a pioneer in the conception and implementation of tandem mass spectrometry (MS/MS) and of desorption ionization, especially molecular secondary ionization mass spectrometry (SIMS). In 2015 his lab performed exploratory analysis of small molecules in cerebrospinal fluid from which the MRM-profiling method emerged. His work also includes the development of miniature portable mass spectrometers using ambient ionization and application of this combination to problems of trace chemical analysis at point-of-care. His interests in the fundamentals of ion chemistry focus on chiral analysis based on the kinetics of cluster ion fragmentation. His group also studies collisions of ions at surfaces for new methods of molecular surface tailoring and analysis, and nanomaterials preparation by soft-landing of ions and charged droplets. Dr. Cooks also launched new method of preparative mass spectrometry based on accelerated reactions in microdroplets. Dr. Cooks has been recognized with the Mass Spectrometry and the Analytical Chemistry awards of the American Chemical Society, the Robert Boyle Medal and the Centennial Prize of the Royal Society of Chemistry, and the Camille & Henry Dreyfus Prize in the Chemical Sciences. He is an elected fellow of the American Academy of Arts and Sciences, the Academy of Inventors and the U.S. National Academy of Sciences.

In this talk, we invite the audience to critically investigate the predominant mass spectrometry workflows used for the exploratory analysis of small molecules and discuss its analytical aspects. We will then present an approach for exploratory lipidomics and metabolomics, multiple reaction monitoring (MRM)-profiling, which initially explores small molecules present in biological samples based on their chemical functionalities using precursor ion (Prec) and neutral loss (NL) scans without the use of chromatographic separation. In a second step the Prec and NL information is translated into MRM scans and these are used to obtain mass profiles to be compared among samples by univariate and multivariate statistical methods. The MRM profiling methodology is characterized by high speed and excellent classification of samples. Identification and quantitation of individual molecules is also achieved.

*This talk was originally scheduled to be presented at MSACL 2020 US by John Yates. It is expected to be about 20 minutes, not including Q&A.

INTRODUCTION: Proteins have exquisite three-dimensional structure. Their structures dictate the functions and interactions within the cell. Most of what we know about the structures of proteins comes from methods like x-ray crystallography, NMR and more recently cyro-EM. These methods are all in vitro methods that often look at proteins as single entities or as a protein complex. What is often missing is the in vivo context to the protein structure, e.g. what is the structure that exists in the cell. Several mass spectrometry-based methods are emerging to examine the 3D proteome or the conformations of proteins in their in vivo context.

OBJECTIVES: The primary objective of this work is to develop methods to measure the "structures" or confirmations of protein in the in vivo environment and to apply them to common diseases.

METHODS: We’ve developed a strategy to measure the surface accessibility of proteins in vivo that provides information about protein conformations and does so in a quantitative manner.

RESULTS: This method has been used to study protein misfolding diseases. This method has been applied to WT and mutant DF508 CFTR to examine structural changes induced by the mutation. A significant change has been observed at a critical interface between the NBD1 and NBD2 domains. This method is also being applied in Alzheimer's disease (AD)and Lewy body disease (LBD) to measure extent of conformational changes to proteins. This method is applied to AD and LBD patient brain tissue lysates and extensive protein comformational changes are observed.

CONCLUSIONS: Substantial changes beyond misfolded aBeta and Tau are observed and based on the molecular changes observed it is difficult to distinguish at the molecular level between advanced AD and LBD suggesting in neurodegenerative misfolding diseases the collapse of proteostasis has a similar endpoint. The studies of CFTR show a critical interface is disrupted between WT and DF508.

I am an analytical chemist focused on infection metallomics and clinical mass spectrometry (MS). From 1991 I have had the main contract with the Institute of Microbiology in Prague, where I run an analytical lab with 25 employees now benefitting from mass spectrometry, nuclear magnetic resonance spectroscopy, and electron microscopy (https://ms.biomed.cas.cz/). As a professor of analytical chemistry, I am also active at Palacky University in Olomouc, CZ, providing me with access to elemental imaging mass spectrometry and infection models. I have a scientific profile with 3582 citations (access date May 21, 2020), 1517 citations since 2015. My current h-index is 30 (Google Scholar). In 2020 I contributed to 11 peer-reviewed papers, nine of them I corresponded.

*This talk was originally scheduled to be presented at MSACL 2020 US. It is expected to be about 35-45 minutes, not including Q&A.

Background: The detection and quantitation of siderophores in urine represents an innovative early indication of pathogenic microorganisms in a host [1]. In this presentation, we document why the general application of siderophores in invasive infectious diagnostics will soon represent the same clinical breakthrough as ribosomal protein typing ten years ago. In addition to handling co-infections or polymicrobial frenemies, with mass spectrometry, we can monitor the antimicrobial drug metabolism [2].

Methods: A database of 700 microbial siderophores was used for screening a patient’s status with invasive infections caused by Aspergillus fumigatus, Pseudomonas aeruginosa, and zygomycetes. Human urine samples were examined by liquid chromatography and Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer [3]. The biomarkers were quantified against matrix-matched standards, and our in-house tool called CycloBranch (https://ms.biomed.cas.cz/cyclobranch) was used for compound dereplication [4].

Results: For the diagnosis of aspergillosis, we used an array of ferri-triacetylfusarinine C (TAFC)/ferri-ferricrocine (FC)/gliotoxin (GTX) in a cohort of 28 patients (14 positive and 14 negative controls). LOD for TAFC in human urine was 0.1 ng/mL. The maximum experienced concentration was 1.7 ug/mL. In the working dataset of critically ill, mostly non-neutropenic humans, the TAFC/FC/GTX sensitivity in urine was 93% compared to standard GM in serum (36%). The kinetic profiles upon antimycotic treatment showed a quick drop in siderophore production in vivo. In the same cohort, ferri-pyoverdines and pyochelin were detected as urinal biomarkers in the patients with Pseudomonas aeruginosa. The LOD for Fe-PVD in urine was 10 ng/mL. In serum samples, the LOD and LOQs were 1 and 5 ng/mL, respectively. In the seminar I will also show the co-infecting frenemies studied in animal models of bacterial [5] or fungal infections [6].

Conclusion: Urinal samples are analytically attractive due to less chemical background compared to serum and clinically interesting due to non-invasive applications. Optimized sample preparation could soon move siderophore analysis from FTICR equipment to standard MALDI-Biotyper-type labs.

References:

1. Luptáková, D.; Pluhá?ek, T.; Pet?ík, M.; Novák, J.; Palyzová, A.; Sokolová, L.; Škríba, A.; Šedivá, B.; Lemr, K. and Havlí?ek, V. Non-invasive and invasive diagnoses of aspergillosis in a rat model by mass spectrometry. Sci Rep 2017, 7, 16523.

2. Houst, J.; Spizek, J. and Havlicek, V. Antifungal Drugs. Metabolites 2020, 10, article No. 106.

3. Pluhacek, T.; Skriba, A.; Novak, J.; Luptakova, D. and Havlicek, V. Analysis of Microbial Siderophores by Mass Spectrometry. In: SK Bhattacharya (ed) Metabolomics: Methods and Protocols 2019: 131-153.

4. Novák, J.; Škríba, A. and Havlí?ek, V. CycloBranch 2: Molecular Formula Annotations Applied to imzML Data Sets in Bimodal Fusion and LC-MS Data Files. Anal Chem 2020, 92, 6844-6849.

5. Petrik, M.; Umlaufova, E.; Raclavsky, V.; Palyzova, A.; Havlicek, V.; Haas, H.; Novy, Z.; Dolezal, D.; Hajduch, M. and Decristoforo, C. Imaging of Pseudomonas aeruginosa infection with Ga-68 labelled pyoverdine for positron emission tomography. Sci Rep 2018, 8, 15698.

6. Skriba, A.; Pluhacek, T.; Palyzova, A.; Novy, Z.; Lemr, K.; Hajduch, M.; Petrik, M. and Havlicek, V. Early and Non-invasive Diagnosis of Aspergillosis Revealed by Infection Kinetics Monitored in a Rat Model. Front Microbiol 2018, 9, 2356.

David Tabb, born in the U.S. Midwest, surprisingly finds himself living in South Africa after a fifteen-year transition from assistant professor to associate professor to “full” professor. While in the United States, he conducted his research in a state university, a private research institute, a national laboratory, and a private university. Since 2015, he has enjoyed the moderate climate of Cape Town, where he is completing a five-year contract in tuberculosis biomarker research at Stellenbosch University. He has survived and occasionally thrived in the rich world of clinical proteomics, where he has specialized in the informatics of protein identification from tandem mass spectrometry. He was particularly fortunate to be a bioinformatics lead for the Clinical Proteomics Technology Assessment for Cancer (CPTAC) during the first ten years of that program’s operations. His hobbies include choral singing, computer upgrading, board gaming, and cat pestering.

Proteomics is a field in constant demand in academia, industry, and the clinical lab. With the rapidly changing technology in the proteomics world, bioinformaticians who have an intimate understanding of mass spectrometry as well as computational skills are much needed. Join our Fireside Chat with clinical proteomics bioinformatician, Dr. David Tabb to learn more about a career in bioinformatics from all angles.
This session will be interactive and offers opportunities to network.

Michael H. Gelb is Professor of Chemistry and Barbara L. Weinstein Endowed Chair in Chemistry, Adjunct Professor of Biochemistry at the University of Washington. Major developments in the Gelb lab include discovery of protein prenylation, development of ICAT proteomic reagents, identification of phospholipases involved in lipid mediator generation, development of anti-parasite drugs, and development of mass spectrometry for newborn screening. Awards include: Repligen Award in Chemistry of Biological Processes (Amer. Chem. Soc.), Univ.of Washington Faculty Lecture Award, Gustavus John Esselen Award (Harvard Univ.), AAAS Fellow, NIH Merit Award, Medicines for Malaria Project of the Year Award, Pfizer Award in Enzyme Chemistry, ICI Pharmaceuticals Award for Excellence in Chemistry. The Gelb lab has published more than 500 papers and 100 patents in biological chemistry. The Gelb laboratory has developed mass spectrometry for worldwide newborn screening of lysosomal storage diseases (the latest expansion of newborn screening panels).

Mass spectrometry has a strong presence in newborn screening laboratories because of the ability to quantify numerous metabolites in dried blood spots on newborn screening cards. Over the past decade mass spectrometry has been developed to measure enzymatic activities in dried blood spots. We will present an 18-plex mass spectrometry assay that provides enzymatic activities and biomarkers for lysosomal diseases and allows consolidation with existing mass spectrometry newborn screening panels. In the second part of the presentation we will show the feasibility of using mass spectrometry for proteomics-based newborn screening. This allows screening for a panel of treatable diseaes for which no other methods exists.
As more and more inborn errors become treatable, in part due to gene therapy, it will be important to develop a universal screening platform that allows high throughput, consolidated newborn screening. Despite great progress in DNA sequencing technology, this will not replace biochemical newborn screening in the foreseable future.

INTRODUCTION: Cystic Fibrosis is a disease caused by mutations in the cystic fibrosis ion transport regulator gene (CFTR). The most common mutation, deletion of Phe at position 508 (DF508), results in a loss of function phenotype that causes a large number of clinical symptoms including chronic respiratory infections. At the protein level, the DF508 mutation causes the protein to misfold which results in rapid degradation of the protein in the ER.

OBJECTIVES: The objective of this study is to understand the mechanisms involved in regulating protein misfolding to learn how to rescue the misfolded protein. A detailed understanding of the mechanism should lead to the identification of drug targets for treatment of the disease.

METHODS: Mass spectrometry-based proteomics was used to identify the proteins interacting with wild type CFTR and DF508 CFTR to identify those proteins interacting with the mutant protein, but not the wild type. Proteins interacting uniquely with the mutant were knocked down to identify those proteins whose “inhibition” could potentially rescue the misfolded protein. In addition, regulation of the trafficking process by post translational modifications was also determined using mass spectrometry.

RESULTS: A much better understanding of the mechanisms behind rescue of the mutant protein has been derived from these studies. A disease specific interactome was identified. Proteins interacting with DF508 whose knockdown rescue the mutant protein have been identified. A PTM code has been identified that must be present for the protein to exit the ER. In addition, structural details of the misfolded protein have been determined using a surface accessibility method.

CONCLUSION: By understanding the mechanism behind the loss of function phenotype of DF508 CFTR we now have better routes to rescue the protein and treat CF disease. This knowledge can be used to understand other mutations in the gene that also result non-trafficking of the protein.

Dr. Yonghao Yu received his Ph.D. in Chemistry from the University of California, Berkeley in 2006 under the direction of Julie Leary, where he developed mass spectrometric approaches for the study of tyrosine sulfation, a protein post-translational modification that is implicated in regulating protein-protein interactions in the extracellular space. In 2007, Dr. Yu joined the laboratories of Steven Gygi and John Blenis in the Department of Cell Biology at Harvard Medical School for his post-doctoral training. There he developed quantitative mass spectrometric strategies for the study of protein phosphorylation.

In 2012, Dr. Yu began his independent research career as an Assistant Professor in the Department of Biochemistry at UT Southwestern Medical Center. He was promoted to Associate Professor with tenure in 2017. Throughout his career, Dr. Yu has been the recipient of numerous awards for his research, including the Tuberous Sclerosis Alliance Postdoctoral Fellowship, a CPRIT Scholar in Cancer Research award, a Virginia Murchison Linthicum Scholar in Medical Research award, a Research Scholar award from the American Cancer Society, a UT System Rising STARs Award and most recently, an R35 MIRA award from NIGMS. He has served on many NIH and DoD advisory panels, including as a current member of the NIH Enabling Bioanalytical and Imaging Technologies (EBIT) Study Section.

The long-term goals of the Yu lab are to develop cutting-edge, mass spectrometry-based proteomic technologies, and applying them to systematically identify and characterize novel protein modification events implicated in various pathophysiological conditions. These data-driven strategies are then combined with classical biochemistry approaches to identify aberrant protein modification patterns, decipher the mechanisms of their deregulation, establish the functional consequences of these molecular events, facilitate the development of relevant therapeutic strategies, and finally, identify proteomic signatures that may serve as diagnostic, prognostic or predictive biomarkers for the relevant diseases (e.g., cancer, diabetes and neurodegenerative disease).

Our understanding of how the biology of various diseases relates to the central dogma that DNA encodes RNA, which encodes protein has been buoyed by rapid technological advances in DNA and RNA sequencing and has led to some of the first advances in personalized medicine. However, characterization of the final and arguably most actionable element of the central dogma, protein, has lagged behind. We are interested in developing mass spectrometry-based quantitative proteomic technologies for the comprehensive characterization of the proteome, with a particular focus on post-translational modifications (PTMs)

Poly-ADP-ribosylation (PARylation) is a protein posttranslational modification (PTM) that was first documented in 1963. PARylation is catalyzed by a family of enzymes called Poly-ADP-ribose polymerases (PARPs). In particular, PARP1 is a nuclear protein that is activated as a result of sensing DNA strand breaks. The critical roles of PARP1 in mediating DNA repair and also cell death provide the rationale for developing PARP1 inhibitors to treat a number of human diseases, including cancer and ischemia reperfusion injury. Indeed, late-stage clinical studies revealed that PARP1 inhibitor treatment significantly prolonged progression-free survival of BRCA-deficient ovarian and breast cancer patients. This led to the recent FDA approval of four PARP1 inhibitors in these two indications. However, the signaling mechanism of PARP enzymes is poorly understood, because PARylation is a labile and heterogenous modification.

To address these pressing questions, we developed a large-scale mass spectrometric approach towards comprehensive characterization of the Asp- and Glu-PARylated proteome. Remarkably, the modified proteins are involved in not only DNA damage repair, but also a surprisingly wide array of other nuclear functions. Using a quantitative mass spectrometry experiment, we also identified many previously unknown PARP1 downstream targets, whose PARylation is sensitive to clinically relevant PARP1 inhibitors. More recently, we generated a cell-specific atlas for protein PARylation map in breast cancer. In doing so, we identified PARylation signatures that correlated with the selective cytotoxicity of PARP1 inhibitors in certain breast cancer cells. These results provide potential predictive biomarkers for PARP1 inhibitors. Finally, using the PROTAC technology, we recently developed a small molecule compound that selectively induces the degradation of PARP1. This compound is able to inhibit PARP1, without eliciting the deleterious effects of PARP1 trapping. We showed that this “non-trapping” PARP1 degrader protects primary cardiomyocytes from genotoxicity-induced cell death. These compounds therefore provide an ideal approach for the amelioration of the various pathological conditions caused by PARP1 hyperactivation.

We will discuss patient-specific effects, sample collection, transport and handling of samples, and tissue preparation when acquiring and interpreting mass spectrometry data. We will especially focus on how these steps effect imaging mass spectrometry data, but many of these topics translate universally across mass spectrometry approaches in general.

This will be an open forum with predefined topics and panelists to guide the conversation.

this isn't it

My research activities focus on improving existing analytic diagnostic tools and expanding the repertoire of informative biomolecules available for disease detection in children. We have developed novel analytic tools for the detection of inborn errors of metabolism and drug exposure in children and continue to explore the value of broad metabolic profiling in conditions such as pre-eclampsia, intrauterine growth retardation, liver failure, and neonatal hypoglycemia.

Mass spectrometry continues to play a prominent role in the field of laboratory medicine. In this session, each speaker will highlight a clinical case where Mass Spec Analysis has made all the difference in a patient's care and outcome.

After completing this activity, the learner will be able to:
1. site examples where mass spectrometry made a difference in patient care
2. describe how mass spectrometry could play an important in their institutions
3. summarize the role of mass spectrometry played in each of these case studies

Robin oversees all Research &Product Development programs within Metabolomic Diagnostics, a company focused on developing early pregnancy risk stratification tests for obstetrical syndromes like preeclampsia and preterm birth. He trained as an analytical chemist, specialized in mass spectrometry, and received a PhD from the University of Antwerp in 2006. After 7 years with a Belgian proteomics biomarker discovery company, where he led the preeclampsia biomarker discovery program, Robin joined Metabolomic Diagnostics in 2013. Since then, he and his team established a translational research workflow which aims to overcome the challenges of translating promising biomarkers into novel products.

Operating at the interface of translational research and commercialisation, he has always keenly engaged in conversations with members of the clinical research community on how to deliver on a shared mission of improving pregnancy outcomes.

Robin has co-authored 20+ original research articles and is a named inventor on 10 patent applications.

Grégoire is a biostatistician specialised in clinical diagnostics and medical devices. Based on complexity estimations, he implements data analysis strategies and statistical frameworks which mitigate risks and therefore increase the chance of successful outcomes. This is achieved by working at the crossroads of statistics, machine learning, medical sciences, and epidemiology whilst adopting IT solutions which are compliant with regulatory and legal frameworks. As a consultant, Grégoire has an established track record of fruitful collaborations with both academic and industry partners.

The area under the receiver operating characteristic (AUROC) curve is a widely used statistic to evaluate performance of prognostic and diagnostic tests. Because AUROC does not depend on disease prevalence, the statistic has also gained prominence in biomarker discovery and development, as it enables biomarker evaluation using cost-effective case-control study designs.
However, end-users in clinical settings typically assess the merits of prognostic / diagnostic tests in function of patient harm and benefit. Disease prevalence and clinical context are critical determinants in clinical utility evaluations and therefore different statistics, like positive and negative predictive values (PPV and NPV), which account for these determinants are typically used to gauge clinical utility. Albeit most biomarker work is performed with the goal of improving clinical care, clinical utility is rarely considered during test development.

To address this lacuna, we devised a method for plotting PPV or NPV criteria, which account for prevalence, in the receiver operating characteristic (ROC) space. Herewith, test developers and clinical end-users are provided with a common framework to discuss and evaluate prognostic / diagnostic test performances.

In this talk we will briefly review key concepts like Sensitivity (Sn), Specificity (Sp) and Prevalence (Pr), and how they are used to create ROC curves (Sn, Sp) and calculate predictive values (Sn, Sp, Pr). We will highlight the limitations of solely depending on AUROC in test evaluations and the importance of considering the shape of ROC curves. Then we will demonstrate how PPV and NPV criteria can be plotted on the ROC space and how this information can be used to discuss test performance in function of clinical utility with different stakeholders. We will conclude with the demonstration of a freely accessible web-tool developed to allow people to explore the dynamic interplay between the test characteristics Sn, Sp, Pr, AUROC, ROC curve shape, PPV and NPV.

Paper: A novel method for interrogating receiver operating characteristic curves for assessing prognostic tests

Software Tools : Positive and negative predictive values

Hubert W. Vesper, Ph.D., is the director of the Clinical Standardization Programs at the
Centers for Disease Control and Prevention’s (CDC) National Center for Environmental
Health. He leads CDC’s clinical laboratory standardization programs to improve the
diagnosis, treatment, and prevention of selected chronic diseases and oversees and
represents specific biomonitoring programs to assess human exposure to environmental
chemicals and its potential impact on human health. He also is co-chair of the steering
committee of the Partnership for Accuracy in Tests for Hormones (PATH), a member of
the steering committee of the National Glycohemoglobin Standardization Program, and
an adjunct faculty member in the Nutrition and Health Sciences Program at Emory
University. Previously he was a consultant at the Kuwait Institute for Scientific
Research, co-chair of the Cardiovascular Biomarker Standardization Steering
Committee, and a member of the World Health Organization (WHO)/Pan American
Health Organization/CDC Emergency Response Team. He received his B.S. in food
science and M.Sc. in food chemistry from the University of Karlsruhe, Germany, and his
Ph.D. from the Technical University of Munich, Germany

Overview of ongoing and new CDC’s Standardization Programs

EDUCATION:
Graduate School: University of Virginia (Chemistry), Charlottesville, VA
Clinical Chemistry and Laboratory Medicine Fellowship: University of Virginia, Charlottesville, VA

CLINICAL:
Laboratory testing in Clinical Chemistry, Toxicology, Hemostasis, and Endocrinology.

RESEARCH:
Liquid chromatography mass spectrometry assay methods.

Dustin R. Bunch, PhD, DABCC, is the assistant director of Clinical Chemistry and Laboratory Informatics at Nationwide Children's Hospital. He is board certified by The Commission on Accreditation in Clinical Chemistry (ComACC) in clinical chemistry and completed his clinical chemistry fellowship at Yale-New Haven Hospital/Yale University in Jun 2019. He received his PhD in Clinical/Bioanalytical Chemistry from Cleveland State University in 2016 and BA in Biochemistry from Case Western Reserve University in 2004. Over the years his research has focused mainly on development and validation of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods; however, recently he has been performing research on hospital and clinical informatics. He is active in professional organizations (AACC, ASCP, and MSACL) for both the local and national level serving on boards and committees.

Working with R for Validation Summaries in the Clinical Laboratory
Lindsay Bazydlo

Putting R Skills to Use in Clinical Laboratories
Dustin Bunch

Dr. Mark Marzinke is Associate Professor of Pathology and Medicine at the Johns Hopkins University School of Medicine. Dr. Marzinke serves as the Director of General Chemistry in the Johns Hopkins Hospital Core Laboratories, and is the Director of the Clinical Pharmacology Analytical Laboratory within the Division of Clinical Pharmacology. He is also Co-Director of the HIV Prevention Trials Network Laboratory Center.

He received his A.B. from the College of the Holy Cross and earned his Ph.D. at the University of Wisconsin-Madison. He completed a fellowship in clinical chemistry at the Johns Hopkins School of Medicine

An overview of the fundamentals that are driving research and development with respect to Sample Preparation.
This is part of a series of Primers on topics relevant to Clinical Mass Spectrometry.

Learning objectives include:
1. Be able to define what Sample Preparation is, why it matters, and why you personally should care. What is the clinical relevance?
2. Understand how sample prep relates to mass spectrometry.
3. Define any terminology that is specific to Sample Preparation.
4. Describe how it works, what are the methods and workflows used and/or how is it implemented in clinical labs.
5. Identify any challenges to implementation/adoption, where do the opportunities lie?

# Originally scheduled for MSACL2020 US in the Scientific Session : Fentanyl at the Forefront : Using Non-traditional Approaches to Identify Fentanyl Analogs

Introduction

Deaths due to opioid overdoses have been on the rise in the United States, with a particular spike in this trend caused by the emergence of synthetic opioids including fentanyl and analogues. There have been numerous reported cases of synthetic opioids as adulterants in heroin, methamphetamine, cocaine, and counterfeit pills. As a result, they are often times consumed unknowingly, causing mixed toxidromes and confounding diagnosis. Proper routine drug monitoring is essential in addressing the ever-expanding magnitude of the opioid epidemic.

Objectives

The primary objective of this study was to observe prevalence of fentanyl analogs and other false positives in positive fentanyl screens of emergency medicine populations in order to evaluate trends in the dynamic landscape of substance abuse.

Methods

Analysis of remnant clinical samples was approved by the UCSF Institutional Review Board. Urine samples from emergency department (ED) patients with drugs-of-abuse screens performed were monitored from October 2018 to March 2019 for presence of fentanyl (via immunoassay). Samples with a positive fentanyl screen were collected and further characterized by high resolution mass spectrometry (LC-HRMS). Urine samples were diluted 1:5. Chromatography was performed using a Kinetex C18 column with a 10-minute gradient from 2%-100% organic. Data was collected on a SCIEX TripleTOF®5600 using a positive-ion mode TOF-MS survey scan with IDA-triggered collection of high resolution product ion spectra (20 dependent scans). Data was screened for fentanyl, norfentanyl, 13 literature-reported synthetic opioids, and compounds reported/suspected to cause false positives with fentanyl immunoassays.

Results

158 emergency department patient samples screened positive via EMIT fentanyl immunoassay (7.44% of all ED samples screened for drugs of abuse). These samples were analyzed via LC-HRMS and analysis was targeted for 15 synthetic opioids and commonly prescribed or over-the-counter pharmaceuticals with chemical structures similar to that of fentanyl. 6.9% of samples were found to be positive for both fentanyl and acetyl fentanyl. Additionally, risperidone, its metabolite 9-hydroxyrisperidone (paliperidone), and loperamide with its metabolite desmethylloperamide were discovered as sources of false positive for fentanyl screens.

Conclusion

This data reveals the non-trivial prevalence of fentanyl in the ED patient population being screened for drugs of abuse. In many of the cases, fentanyl was not suspected or discovered as exposure agent prior to more comprehensive testing or patient discharge. The identification of acetyl fentanyl along with fentanyl via HRMS analysis can hint to a changing trend in provenance of illicitly manufactured fentanyl. Finally, HRMS is a valuable tool to identify compounds responsible for reoccurring false positives that can inform better patient care and offer a more accurate picture of the continually evolving opioid epidemic.

Robert L. Fitzgerald, PhD, DABCC Dr. Fitzgerald received his BS degree in Chemistry at Loyola College of Maryland, and his PhD in Pharmacology/Toxicology at the Medical College of Virginia/Virginia Commonwealth University. After two and a half years as a forensic toxicologist for the State of Virginia, he took a position as the Director of the Mass Spectrometry Laboratory at the San Diego VA Hospital. Currently, Dr. Fitzgerald is a Professor in the Department of Pathology at the University of California, San Diego where he is the director of the toxicology laboratory and associate director of the clinical chemistry laboratory. He is board certified in toxicology and clinical chemistry by the American Board of Clinical Chemistry. He is the director of the clinical chemistry fellowship at UCSD.

I graduated in analytical chemistry from the faculty of chemistry at Shahid Beheshti University (Tehran,Iran).For my Master's thesis I was working on the Optimization of solid phase microextraction and electromembrane extraction techniques to measure certain pollutants and drugs. During my studies,I was also awarded medals and honorary diplomas from international and national competitions and expositions because of my inventions. In June 2017, I started my Glysign PhD project, conducting research at Genos Ltd., Zagreb, Croatia, and the Leiden University Medical Centre in Leiden, the Netherlands. My research is geared towards the quantification of serum protein glycosylation for patient stratification in early-onset and other diabetes types.

# Originally scheduled to be presented at MSACL 2020 US. Moved from August 10 make-up session due to timing conflict.

Introduction

Type 2 diabetes mellitus (T2D) represents a major public health challenge. The total plasma N-glycome (TPNG) reflects the levels and glycosylation of major plasma glycoproteins, among which are immunoglobulins, acute-phase proteins and apolipoproteins. Although associations of TPNG with T2D have been recently reported by us and others, data on the association between TPNG and T2D complications are scarce. Here, we applied our new ultrahigh-resolution MS method to assess associations of TPNG with T2D complications.

Methods

We determined the TPNG in a subsample of the Hoorn Diabetes Care System cohort (n = 1519 T2D cases and 192 nondiabetic controls from the New Hoorn study cohort) using our Fourier-transform ion cyclotron resonance MS method. Blood plasma samples were randomized over 21 96-well plates, together with technical replicates. Prior to MS analysis, N-glycans were released using PNGase F. For stabilization and linkage differentiation, sialic acids were derivatized. Released glycans were purified and spotted using an automated liquid handling platform. In total, 68 individual glycan compositions were quantified after total-area normalization and summarized into derived traits representing structural features. By applying logistic regression models, we tested for associations of TPNG with case-control status and micro- or macrovascular complications, such as nephropathy, retinopathy, or cardiovascular disease (cross-sectional). T2D complications were also assessed prospectively by Cox regression. Models were adjusted for age, sex, and T2D risk factors. The outputs were meta-analyzed together with our MALDI-TOF-MS data from the independent cohort DiaGene (1815 T2D cases and 836 controls) which we obtained and described previously.

Results

The recently developed method using MALDI-FTICR-MS improved resolution, mass accuracy, dynamic range and the signal-to-noise ratio, especially for larger glycans, in comparison to our previous MALDI-TOF workflow. The relative standard deviation over the 68 detected glycan species was on average 6.6%, based on the relative intensities from 93 technical replicates of pooled plasma, representing total method repeatability.

We found significant associations of all major glycosylation traits, i.e. sialylation, fucosylation, galactosylation, bisection, and glycan complexity with T2D in the meta-analyses of both cross-sectional and prospective data. For example, the bisection and galactosylation of immunoglobulin-related glycans were associated with macrovascular complications and nephropathy. We aim to confirm these results in the near future on protein-specific level, by isolating IgG prior to released-glycan analysis.

Conclusions

Our new method improved the detection of several glycan species, thus providing possible new insights into the association between TPNG and T2D. Our results will be taken forward to improve personalized approaches in T2D and related complications in the future.

Dr. Robert D. Voyksner received his B.S. in Chemistry at Canisius College in 1978 and his Ph.D. at the University of North Carolina at Chapel Hill in 1982.

As a member of the Research Triangle Institute staff since 1983, Robert has been responsible for developing extraction, separation and mass spectrometric methods for biologically and environmentally significant compounds. He has primarily employed HPLC/MS on quadrupoles, sectors and ion trap mass spectrometers for positive and negative ion detection for specific compounds. His pioneering work in HPLC/MS and HPLC/ion trap MS has demonstrated the capability to perform analysis of compounds not amenable to analysis by GC/MS. Specifically, he has worked on the development and evaluation of electrospray HPLC/MS methods for the analysis of high molecular weight opioid peptides, antimalarials, anticancer drugs, nucleotides, antibiotics, proteins and cyclodextrins. He has been working with FDA for the past nine years to develop new methodology using electrospray HPLC/MS and capillary electrophoresis-MS to detect and measure antibiotics (-lactams and aminoglycosides) in food and milk to insure human food safety.
In addition, he is has developed methods and evaluated the use of electrospray HPLC/MS and ion trap MS/MS for environmental monitoring, particularly for the detection of azo dyes and pesticides (including carbamate, methyl urea, triazine, organophosphorous and phenolic acid ). He has developed CE/MS and CE/Ion trap MS methods for the identification of DNA adducts from exposure to nitrocompounds and pesticides.

Dr. Voyksner's research in mass spectrometry has resulted in over 230 publications and presentations, primarily in the area of HPLC/MS. He has served on the Board of The American Society For Mass Spectrometry (ASMS), is on the organization committee for The Montreux LC/MS Symposium, and was the organizer for the 1995, 1999 and 2003 Montreux LC/MS Symposium. He is the organizer for the 2007 Montreux LC/MS Symposium (location yet to be determined). Dr. Voyksner has taught LC/MS short courses for the past 15 years for ASMS, every major pharmaceutical company, ISSX, PBA, HPCE and other HPLC focused meetings.

Now, as president of LCMS Limited, Robert has brought his expertise in mass spectrometry along with years of experience in training and problem solving together into focus and guides LCMS Limited to strive for excellence in its mission of "...chasing tomorrow through research, innovation and education today....."

This 4-day ONLINE ONLY course is designed for the chromatographer / mass spectrometrist who wants to be successful in developing methods, method optimization and solving problems using LC-MS/MS. The course covers the atmospheric pressure ionization (API) techniques of electrospray, pneumatically assisted electrospray and atmospheric pressure chemical ionization (APCI) and atmospheric pressure photo ionization (APPI) using single quadrupole, triple quadrupole, time-of-flight and ion trap mass analyzers.

Discussions of sample preparation and chromatography will target method development and optimization for the analysis of "real-world" samples by LC/MS/MS.

The course highlights the following topics with respect to optimization a method to achieve the best sensitivity, specificity and sample throughput:

1. Optimization ionization in API techniques,
2. Understanding and minimizing matrix suppression,
3. Relative merits of various LC column lengths, particle sizes and column diameters for LC/MS/MS analysis,
4. Introduction into the interpretation of MS/MS spectra,
5. Important issues in LC/MS/MS quantitation, and
6. Optimization of an quantitative analysis.

Applications of LC/MS/MS to analyze compounds of clinical interest in biological matrices will be discussed throughout the course to emphasize the topics covered.

John A. McLean is Stevenson Professor of Chemistry, Chair of the Department of Chemistry, Past Chair of the Faculty Senate, Director of the Center for Innovative Technologies, Deputy Director of the Institute for Integrative Biosystems Research and Education at Vanderbilt University. He received his Ph.D. from George Washington University, was a postdoctoral fellow at Forschungszentrum Jülich in Germany with Prof. Dr. Sabine Becker, and a postdoctoral at Texas A&M University with Prof. David H. Russell before joining the Vanderbilt Faculty as an assistant professor in 2006. McLean and colleagues focus on the conceptualization, design, and construction of structural mass spectrometers, specifically targeting complex samples in systems, synthetic, and chemical biology. His group applies these strategies to forefront translational research areas in drug discovery, precision medicine, and ‘human-on-chip’ synthetic biology platforms. He served on the board of directors for the American Society for Mass Spectrometry and serves in an editorial role on the boards of several leading scientific journals. He has been honored to work closely with the NAS and NSF in several roles, including recently co-authoring an NSF report on big data science in chemistry. He has received many professional and teaching awards including his laboratory being designated as a Waters Center of Innovation and an Agilent Thought Leader Laboratory for their work in ion mobility-mass spectrometry and translational biosciences. He has published over 150 manuscripts and 30 patents in the areas of innovative bioanalytical chemistry and quantitative sciences.

One of the predominant challenges in systems-wide analyses and molecular phenomics is the broad-scale characterization of the molecular inventory in cells, tissues, and biological fluids. Advances in computational systems biology rely heavily on the experimental capacity to make omics measurements, i.e. integrated metabolomics, proteomics, lipidomics, glycomics, etc., accompanied with fast minimal sample preparation, fast measurements, high concentration dynamic range, low limits of detection, and high selectivity. This confluence of figures-of-merit place demanding challenges on analytical platforms for such analyses. Ion mobility-mass spectrometry (IM-MS) provides rapid (ms) gas-phase electrophoretic separations on the basis of molecular structure and is well suited for integration with rapid (us) mass spectrometry detection techniques. This report will describe recent advances in IM-MS integrated omics measurement strategies in the analyses of complex biological samples of interest in systems, synthetic, and chemical biology in clinical chemistry applications. New advances in bioinformatics and biostatistics will also be described to approach biological queries from an unbiased and untargeted perspective and to quickly mine the massive datasets gathered to provide targeted and actionable information.

Professor Hewison is currently Professor of Molecular Endocrinology in the Institute of Metabolism and Systems Research (IMSR) at The University of Birmingham. He gained his PhD in Biochemistry from Guy’s Hospital Medical School London and then spent nine years at University College London. He then moved to the University of Birmingham where he established the UK’s major vitamin D research group, leading to an appointment as Professor of Molecular Endocrinology in 2004. In 2005 he joined Cedars-Sinai Medical Center Los Angeles but was then recruited to neighbouring UCLA at the end of 2007 as Professor-in-Residence.In September 2014 Professor Hewison returned to The University of Birmingham to join the newly-established IMSR as Deputy Director.

Professor Hewison has published over 200 peer-reviewed manuscripts focused on various facets of steroid hormone endocrinology, and he is Chair of the Vitamin D Workshop (http://www.vitamindworkshop.org/). He currently has a team of two postdocs, four PhD students and a clinical fellow who are funded by US and UK research grants.

Since starting his postdoc work at UCL London in 1986, Dr Hewison has established a series of studies to investigate the role of vitamin D in immune health and disease. This included the first descriptions of vitamin D metabolism as a feature of normal innate immune function, antigen presentation, and antimicrobial immunity. Following a nine year period at UCLA, Los Angeles, Dr Hewison is now back in the UK at the University of Birmingham where his current interests include the role of cell metabolism in immune responses to vitamin D, and the development of new Liquid Chromatography Mass Spectrometry strategies to better determine vitamin D ‘status’.