MSACL 2023 Abstract

INTRODUCTION: Telehealth, accessing healthcare and wellness remotely, is an expanding necessity influenced by overcoming a global pandemic, fluidity of technology, and an increase in chronic diseases from an aging population compounded by poor nutrition and sedentary lifestyles. A diverse and ailing global population has accelerated interest in precision medicine to identify the mechanisms of an individual’s disease state(s) so that an effective intervention or treatment can be prescribed to correct or alleviate phenotypic ailments. There has also been a rise in precision health, where individuals are focused on disease prevention, optimizing their quality of life as well as prioritizing and monitoring their health through an array of strategies involving nutrition, physical fitness, healthy microbiome, wearable technology, and mental health wellbeing. The blood proteome is an informative source of biomarkers representing an individual's physiological phenotype or biosignature. The proteome also represents an individual's genetic predisposition, continuously responding to environmental factors, as well as infectious agents, physical exercise and nutritional intervention. The convenience of having a robust remote collection device for blood tests coupled with a large multiplex protein assay representing an individual's biosignature will facilitate access to both precision medicine and precision health. Herein, we tested a 60 protein health surveillance panel (HSP), containing 35 FDA/LDT assays and covering at least 14 pathological states, on 8 healthy individuals' ability to collect their own capillary blood from a lancet finger prick onto remote collection Mitra devices (n=6) and directly compared this to the traditional phlebotomist venous blood draw also placed on Mitra devices (n=6) and plasma collection method (n=3).

METHODS: All samples from the 8 individuals were spiked with stable-isotope-labelled (SIL) HSP peptides and quantitatively analyzed by liquid chromatography-multiple reaction monitoring-mass spectrometry (LC/MRM-MS) scheduled method targeting 466 ion transitions, 114 peptides representing the 60 HSP proteins and by a data-independent acquisition mass spectrometry (DIA-MS) discovery method. Four sets of quality control (QC) groups were utilized: pooled from the digestion of capillary blood of all 8 volunteers biological replicates (n=48), pooled from the digestion of venous blood of all 8 volunteers biological replicates (n=48), pooled from the digestion of plasma of all 8 volunteers (isolated from venous blood) biological replicates (n=24), and a purchased human pooled plasma (pool4) used at the start, middle and end of each sample plate analysis. The QC samples from two sample plates were analyzed on two harmonized LC/MRM-MS platforms consisting of a Thermo Ultimate 3000 HPLC and Sciex 6500+ triple quadrupole mass spectrometer which resulted in an average %CV for all quantifier transitions of <11% for capillary blood, <6% venous blood, and <10% plasma.

RESULTS: For simplicity, we compared the 8 individuals and their biological replicates average peak area ratios (PAR) of the quantifier peptide transitions of all HSP proteins between the capillary blood (n=48), venous blood (n=48) and matched plasma (n=24) and all were below 20% CV. Heat map analysis of all 8 volunteers demonstrated that each individual had a unique biosignature and that three of the volunteers had an increase signal in inflammatory proteins including C-reactive protein (CRP). Hierarchical analysis of the 8 volunteers and their 6 biological replicates clustered in both their capillary blood and venous blood. Biological replicates from capillary blood and venous blood clustered for the same volunteer in k-means clustering analysis. Pearson statistical analysis of the three biofluids indicated that there was >90% similarity suggesting that remote collection devices may be a viable option for personal blood proteome biosignature stratification and health analysis. The same samples were also analyzed by DIA-MS on a 60 minute gradient using a Thermo Ultimate 3000 HPLC interfaced with a Thermo Orbitrap Exploris 480 mass spectrometer, using both a plasma spectral library (all protein identifications totaled 1121, with 399 in venous, 396 in capillary, and 388 in plasma, observed in >66% of samples) and a pan-human spectral library (all protein identifications totaled 3811, with 1029 in venous, 877 in capillary, and 364 in plasma, observed in >66% of samples with 1% FDR). The majority of HSP proteins were also identified in the DIA-MS method, in addition to many other proteins not typically seen in plasma alone that have biological importance in oxidative stress, longevity, energy metabolism, cardiovascular health, cancer, etc. The identified proteins with CV<30% were summed for each individual volunteer and were shown to reproducibly quantitate approximately 600-700 proteins in capillary blood, ~800 proteins in venous blood and ~300-400 proteins in plasma.

CONCLUSION: This study demonstrated that all three biofluids, but more specifically whole blood from a remote collection device, could be used for targeted HSP LC/MRM-MS and DIA-MS relative quantitative discovery analysis with high reproducibility. The DIA-MS results also demonstrates the potential for the development of a multiplexed protein biomarker assay targeting hundreds to possibly over a thousand proteins to capture a comprehensive stratification of individual biosignatures that would substantially inform precision medicine and precision health decisions. The convenience of a remote collection device to patients, the array of circulating protein tests and sample processing efficiency on a Mitra device in addition to the technological advancements in mass spectrometry and bioinformatic tools to quantitively identify thousands of physiologically relevant proteins in whole blood may shift the paradigm away from analyzing plasma alone.