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

Despite huge efforts to map the human proteome using mass spectrometry the overall sequence coverage achieved to date is still below %. 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 ( vs ; [1]% protein FDR). However, subtilisin considerably improved the coverage of missing and particularly proline-rich areas of the proteome. Along [8] high confidence phosphorylation sites identified in total, only % were shared between both enzymes, while % were exclusive to subtilisin. Notably, [6] 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. [ABSTRACT FROM AUTHOR]