Digestion and depletion of abundant proteins improves proteomic coverage

Abstract

Two major challenges in proteomics are the large number of proteins and their broad dynamic range within the cell. We exploited the abundance-dependent Michaelis-Menten kinetics of trypsin digestion to selectively digest and deplete abundant proteins with a method we call DigDeAPr. We validated the depletion mechanism with known yeast protein abundances and observed greater than 3-fold improvement in low abundance human protein identification and quantitation metrics. This methodology should be broadly applicable to many organisms, proteases, and proteomic pipelines.

Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics

“…Pronounced problems are: serious unpredictable protein loss to aggregation (14), high oxidation artifacts and contaminations introduced by laborious processing (15), biases over protease specificity and abundance (13,16), low digestion efficiency, coverage, and peptide reproducibility (2,4).…”

Targeted mass spectrometry analysis of the proteins IGF1, IGF2, IBP2, IBP3 and A2GL by blood protein precipitation

“…This problem is further aggravated when employing fast LC methods for high-throughput analysis that have reduced capacity for analyte separation and therefore higher signal suppression and matrix effects. Thus, the development of simplified and optimised blood sample preparation protocols prior to LC-MS/ MS analysis remains a highly active area of research [9][10][11][12][13][14][15][16].…”

Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias

“…Conventional digestion paradigm subjects membrane proteins to harsh denaturants, repeated separations via precipitations or dial-filtrations, and multiple proteases for day (s). Yet it suffers: low (sub-50%) digestion efficiency (25,26), bias in protein abundance (27), bias in protease specificity sites (28), low peptide reproducibility (27,29), low (sub-30%) sequence coverage (30), high oxidation artifacts, and high contamination (31). Current gel-free digestion methods of purified GABA A R take 2 days combining 6 (4 overnight) digestions (29,32), only able to identify two glycosylation sites (29).…”

“…Current gel-free digestion methods of purified GABA A R take 2 days combining 6 (4 overnight) digestions (29,32), only able to identify two glycosylation sites (29). Representative peptide reproducibility in global shotgun analysis is 25 Ϯ 2%, and 15 Ϯ 2% after DigDeAPr treatment (27), whereas membrane proteins are considered mission impossible. These limitations severely undermine quantitation and preclude HDX or oxidation label-based structural studies.…”

“…These limitations severely undermine quantitation and preclude HDX or oxidation label-based structural studies. Protein abundance bias arises when abundant proteins occupy most protease catalytic sites leaving few available for low-abundance proteins (27); intricate structures and bulky modifications-such as disulfide bonds, glycosylations, and associated lipids, detergents or other proteins-obstruct protease access (25), together pinpointing inadequate enzyme-protein substrate contact as the cause.…”