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Accelerating Proteomics Using Broad Specificity Proteases
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DOI：10.1021/acs.jproteome.3c00852.png)
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Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000x less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required unspecific searches. In-depth analyses of proteinase K, subtilisin, and thermolysin Jurkat digests identified 7374, 8178, and 8753 unique proteins with average sequence coverages of 21, 29, and 37%, including 10,000s of amino acids not reported in PeptideAtlas' >2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products. Finally, proteinase K, subtilisin, and thermolysin enabled label-free quantitation of 3111, 3659, and 4196 unique Jurkat proteins, which in our hands is comparable to trypsin. Our data demonstrate that broad-specificity proteases enable quantitative proteomics of uncharted areas of the proteome. Their fast kinetics may allow on-the-fly digestion of samples in the future.
Keyword:
proteases
bottom-up proteomics
unspecific
nonspecific
high throughput
elastin
pepsin
chymotrypsin
PTM
phosphorylation
期刊
IF:
3.6
论文数:
9.4K
被引数:
2.3W
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