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

Abstract

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.

Label-Free Quantitative Phosphoproteomics of the Fission Yeast Schizosaccharomyces pombe Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies

“…The use of a relatively high concentration of trypsin was shown to compensate for the reduced trypsin digestion efficiency in the proximity of the phosphorylated amino acid residues [35,36]. Alternatively, various proteases, such as Lys-C, Glu-C, Arg-C, Asp-N, Lys-N, chymotrypsin, or subtilisin, might be used for sequential digestion to improve the phosphoproteomics sampling depth [37][38][39]. As have been shown previously, using alternatives to tryptic digestion would enable the detection of phosphopeptides that stayed inaccessible by the trypsin-only digestion [40,41].…”

Targeted and Untargeted Proteomics Approaches in Biomarker Development

“…Despite these technological advancements, discovery proteomics workflows often do not account for PTMs, particularly PTMs other than the most abundant ones, such as phosphorylation or glycosylation. Nearly 200 other PTMs of known biological relevance are typically neglected . Of particular importance in the field of cancer immuno‐oncology is protein glycosylation, which is thought to play an essential role in tumor immune evasion and thus a tumor's resistance to cancer immunotherapeutic intervention .…”

Proteogenomics of Colorectal Cancer Liver Metastases: Complementing Precision Oncology with Phenotypic Data

“…The dried 349-µg aliquot was re-solubilized in 1 mL of 80% ACN, 5% TFA, and 1 M glycolic acid (buffer 1) for phosphopeptide enrichment using titanium dioxide (TiO 2 ; Titansphere TiO, 5 µm particle size, GL Sciences Inc, Japan), as described previously [65,66]. (1) TiO 2 beads (6:1 TiO 2 :peptide, w:w) were added to the sample, followed by incubation for 10 min on a shaker at room temperature.…”