Analyzing the proteome, N-glycoproteome and phosphoproteome of dried blood: method optimization and application to phenotyping of human sepsis

Introduction: Blood is a common matrix for large-scale phenotyping of disease. However, most proteomic studies are restricted to blood plasma, which can suffer from pre-analytical variability and may not capture relevant cellular pathobiology. We are exploring the utility of dried whole blood, collected by volumetric microsampling, for characterization of sepsis. Method development has focused on facile sample collection and storage, plate-based sample prep, and medium- to high-throughput LC-MS/MS analyses of proteins and post-translational modifications.

Methods: For prospective sample collection, twenty microliters of venous whole blood was captured on Neoteryx Mitra devices, dried and transferred to Matrix tubes for -80 °C storage and subsequent processing. Mitra tips were subjected to deoxycholate-assisted trypsin digestion, and ~1 mg of digests were sequentially enriched for N-glycopeptides and phosphopeptides using tip-based hydrophilic interaction chromatography (HILIC) and magnetic nanoparticle-based immobilized metal affinity chromatography (IMAC), respectively. Analysis of unenriched digests used microflow-LC (Waters ACQUITY Premier 1 mm CSH C18) and DIA-MS/MS (Exploris 480), and N-glyco- and phospho-peptide enriched fractions utilized an Evosep One LC and 60 sample-per-day method, and stepped-collision energy DDA-MS/MS (Exploris 480) and DIA-MS/MS (Orbitral Astral), respectively. Spectronaut, Glyco-Decipher and Byonic/Proteome Discoverer were utilized to identify and quantify proteins and PTMs.

Preliminary Results: We developed a streamlined workflow for plate-based processing of dried whole blood for bottom-up proteomics, N-glycoproteomics and phosphoproteomics that avoids sample cleanup or drying steps and can be completed in under 2 days. An optimized phosphoproteomic workflow utilized depletion of glycopeptides, magnetic nanoparticle-based enrichment and DIA using an Orbitrap Astral MS. In less than 2h of total analysis time per sample, approximately 1,100 proteins, 750 glycopeptides, and 6,000 site-localized phosphopeptide precursors were quantified in blood from healthy controls after filtering on a coefficient of variation of <20% in replicates of a study-specific pool. As proof of concept, samples were collected in Tanzania as part of an ongoing NIH-funded sepsis subtyping study. The analyses of these samples and integration with clinical and "omic data is in progress.

Conclusions: A workflow has been developed for comprehensive and precise proteomic profiling of dried whole blood which utilizes minimal sample prep steps and relatively high-throughput LC-MS/MS analysis. Sample collection is easily integrating into typical bio-banking protocols. This overall strategy should enable the discovery of new insights into human sepsis.