Quantifying Missing (Phospho)Proteome Regions with the Broad-Specificity Protease Subtilisin

Humberto Gonczarowska-Jorge, Stefan Loroch, Margherita Dell'Aica, Albert Sickmann, Andreas Roos, René P. Zahedi*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)


Despite huge efforts to map the human proteome using mass spectrometry the overall sequence coverage achieved to date is still below 50%. Reasons for missing areas of the proteome comprise protease-resistant domains including the lack/excess of enzymatic cleavage sites, nonunique peptide sequences, impaired peptide ionization/separation and low expression levels. To access novel areas of the proteome the beneficial use of enzymes complementary to trypsin, such as Glu-C, Asp-N, Lys-N, Arg-C, LysargiNase has been reported. Here, we present how the broad-specificity protease subtilisin enables mapping of previously hidden areas of the proteome. We systematically evaluated its digestion efficiency and reproducibility and compared it to the gold standard in the field, trypsin. Notably, subtilisin allows reproducible near-complete digestion of cells lysates in 1-5 min. As expected from its broad specificity the generation of overlapping peptide sequences reduces the number of identified proteins compared to trypsin (8363 vs 6807; 1% protein FDR). However, subtilisin considerably improved the coverage of missing and particularly proline-rich areas of the proteome. Along 14:628 high confidence phosphorylation sites identified in total, only 33% were shared between both enzymes, while 37% were exclusive to subtilisin. Notably, 926 of these were not even accessible by additional in silico digestion with either Asp-N, Arg-C, Glu-C, Lys-C, or Lys-N. Thus, subtilisin might be particularly beneficial for system-wide profiling of post-translational modification sites. Finally, we demonstrate that subtilisin can be used for reporter-ion based in-depth quantification, providing a precision comparable to trypsin - despite broad specificity and fast digestion that may increase technical variance.

Original languageEnglish
Pages (from-to)13137-13145
Number of pages9
JournalAnalytical Chemistry
Issue number24
Early online date14 Nov 2017
Publication statusPublished - 19 Dec 2017

Bibliographical note

This study was supported by the Ministerium für Kultur und Wissenschaft des Landes Nordrhein-Westfalen, the Senatsverwaltung für Wirtschaft, Technologie und Forschung des Landes Berlin, and the Bundesministerium für Bildung und Forschung. H.G.J. further thanks the CAPES Foundation for financial support. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD008068.(6)
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b02395.


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