Protein digestion using a dedicated protease represents a key element in a typical mass spectrometry (MS)-based shotgun proteomics experiment. Up to now, digestion has been predominantly performed with trypsin, mainly because of its high specificity, widespread availability and ease of use. Lately, it has become apparent that the sole use of trypsin in bottom-up proteomics may impose certain limits in our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments or even subsets of proteins. To overcome this problem, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated from these alternative proteases, have not been systematically documented. Therefore, here we provide an optimized protocol for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, LysC, LysN, AspN, GluC and ArgC. This protocol is formulated to promote ease of use and robustness, which enable parallel digestion with each of the six tested proteases. We present data on protease availability and usage including recommendations for reagent preparation. We additionally describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in ∼2 d.
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This work has been supported by the Netherlands Proteomics Centre, the Netherlands Organization for Scientific Research (NWO) supporting the Roadmap embedded large-scale proteomics facility Proteins@Work (project 184.032.201) and by the PRIME-XS project grant agreement number 262067 supported by the European Community's Seventh Framework Programme (FP7/2007-2013) to AJRH. LT was supported by EMBO with a long-term fellowship (ALTF 776-2013).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Physicochemical characteristics of the peptides obtained by in-silico and experimental digestion of the E. coli proteome
Plots representing profiles of physicochemical characteristics of the peptides obtained by in-silico (left) and experimental (right) digestion of the E. Coli proteome. The properties shown are (a) number of acidic residues, (b) number of aliphatic residues, (c) number of aromatic residues, (d) number of basic residues, (e) hydrophobicity (GRAVY score), (f) peptide length, (g) pI, and (h) number of small residues. Analyses were performed using the R Statistical Programming Language (http://www.r-project.org) package ‘Peptides’.
Supplementary Figure 1 (PDF 303 kb)
Recommended digestion conditions, availability and purity of the here used proteases. (XLSX 14 kb)
Typical elution profile of tryptic BSA peptides (20 fmole injection) during a 45min chromatographic gradient. (XLSX 9 kb)
List of BSA peptides identified in each proteolytic digest and their benchmark against theoretical digestion. (XLSX 52 kb)
List of E. coli proteins and peptides identifications in each proteolytic digest using the enzyme-specific search settings. (XLSX 46899 kb)
List of E. coli proteins and peptides identifications in each proteolytic digest using the non-specific search settings. (XLSX 58541 kb)
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Giansanti, P., Tsiatsiani, L., Low, T. et al. Six alternative proteases for mass spectrometry–based proteomics beyond trypsin. Nat Protoc 11, 993–1006 (2016). https://doi.org/10.1038/nprot.2016.057
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