1.
Small-protein Enrichment Assay Enables the Rapid, Unbiased Analysis of Over 100 Low Abundance Factors from Human Plasma.
Harney, DJ, Hutchison, AT, Su, Z, Hatchwell, L, Heilbronn, LK, Hocking, S, James, DE, Larance, M
Molecular & cellular proteomics : MCP. 2019;(9):1899-1915
Abstract
Unbiased and sensitive quantification of low abundance small proteins in human plasma (e.g. hormones, immune factors, metabolic regulators) remains an unmet need. These small protein factors are typically analyzed individually and using antibodies that can lack specificity. Mass spectrometry (MS)-based proteomics has the potential to address these problems, however the analysis of plasma by MS is plagued by the extremely large dynamic range of this body fluid, with protein abundances spanning at least 13 orders of magnitude. Here we describe an enrichment assay (SPEA), that greatly simplifies the plasma dynamic range problem by enriching small-proteins of 2-10 kDa, enabling the rapid, specific and sensitive quantification of >100 small-protein factors in a single untargeted LC-MS/MS acquisition. Applying this method to perform deep-proteome profiling of human plasma we identify C5ORF46 as a previously uncharacterized human plasma protein. We further demonstrate the reproducibility of our workflow for low abundance protein analysis using a stable-isotope labeled protein standard of insulin spiked into human plasma. SPEA provides the ability to study numerous important hormones in a single rapid assay, which we applied to study the intermittent fasting response and observed several unexpected changes including decreased plasma abundance of the iron homeostasis regulator hepcidin.
2.
N-Terminomics/TAILS Profiling of Proteases and Their Substrates in Ulcerative Colitis.
Gordon, MH, Anowai, A, Young, D, Das, N, Campden, RI, Sekhon, H, Myers, Z, Mainoli, B, Chopra, S, Thuy-Boun, PS, et al
ACS chemical biology. 2019;(11):2471-2483
Abstract
Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1, and catenin delta-1), and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing, or increased remodeling of adherens junctions and other cellular functions. Analysis of the preferred proteolytic cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.