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Broad-specificity enzymes enable fast and cost-effective digestion for in-depth proteomics and precise label free quantitation

ABSTRACT: We have devised 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, thermolysin Jurkat digests identified 7374, 8178, 8752 unique proteins with average sequence coverages of 21%, 29%, 37%, including tens of thousands of amino acids not reported in the PeptideAtlas database spanning over 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. 2D runs: 20 high-pH RP-HPLC fractions analyzed by DDA LC-MS/MS mode on a timsTOF Pro2, with Evosep; 15 SPD method, 200 ng peptide loaded on-column. STRAP vs SP3: each digest prepared in triplicate with SP3 and STRAP, respectiveyl-- analyzed by DDA nano-LC-MS/MS on an Orbitrap Exploris 480, 87 min gradient, 750 ng peptide loaded on-column.

INSTRUMENT(S): timsTOF Pro 2, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Rene Zahedi

PROVIDER: MSV000093532 | MassIVE | Wed Nov 29 05:15:00 GMT 2023

REPOSITORIES: MassIVE

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