posted on 2024-03-08, 20:37authored byXuehui Jiang, Darien Yeung, Yang Liu, Victor Spicer, Havva Afshari, Ying Lao, Francis Lin, Oleg Krokhin, René P. Zahedi
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 200–1000× 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.