Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Six alternative proteases for mass spectrometry–based proteomics beyond trypsin

Abstract

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.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: LC-MS analysis of 20 fmol of BSA digests.
Figure 2: LC-MS analysis of E. coli lysate digests.
Figure 3: Specificity and trend for missed cleavages for each of the six enzymes using the protocol presented here for the digestion of an E. coli lysate.

References

  1. Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).

    CAS  Article  Google Scholar 

  2. de Godoy, L.M.F. et al. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast. Nature 455, 1251–1254 (2008).

    CAS  Article  Google Scholar 

  3. Kim, M.-S. et al. A draft map of the human proteome. Nature 509, 575–581 (2014).

    CAS  Article  Google Scholar 

  4. Wilhelm, M. et al. Mass-spectrometry-based draft of the human proteome. Nature 509, 582–587 (2014).

    CAS  Article  Google Scholar 

  5. Link, A.J. et al. Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682 (1999).

    CAS  Article  Google Scholar 

  6. Wolters, D.A., Washburn, M.P. & Yates, J.R. An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73, 5683–5690 (2001).

    CAS  Article  Google Scholar 

  7. Altelaar, A.F.M., Munoz, J. & Heck, A.J.R. Next-generation proteomics: towards an integrative view of proteome dynamics. Nat. Rev. Genet. 14, 35–48 (2013).

    CAS  Article  Google Scholar 

  8. Yates, J.R., Ruse, C.I. & Nakorchevsky, A. Proteomics by mass spectrometry: approaches, advances, and applications. Annu. Rev. Biomed. Eng. 11, 49–79 (2009).

    CAS  Article  Google Scholar 

  9. Bensimon, A., Heck, A.J.R. & Aebersold, R. Mass spectrometry-based proteomics and network biology. Annu. Rev. Biochem. 81, 379–405 (2012).

    CAS  Article  Google Scholar 

  10. Aebersold, R. & Mann, M. Mass spectrometry-based proteomics. Nature 422, 198–207 (2003).

    CAS  Article  Google Scholar 

  11. Walther, T.C. & Mann, M. Mass spectrometry-based proteomics in cell biology. J. Cell Biol. 190, 491–500 (2010).

    CAS  Article  Google Scholar 

  12. Tsiatsiani, L. & Heck, A.J.R. Proteomics beyond trypsin. FEBS J. 282, 2612–2626 (2015).

    CAS  Article  Google Scholar 

  13. Guo, X., Trudgian, D.C., Lemoff, A., Yadavalli, S. & Mirzaei, H. Confetti: a multiprotease map of the HeLa proteome for comprehensive proteomics. Mol. Cell. Proteomics 13, 1573–1584 (2014).

    CAS  Article  Google Scholar 

  14. Swaney, D.L., Wenger, C.D. & Coon, J.J. Value of using multiple proteases for large-scale mass spectrometry-based proteomics. J. Proteome Res. 9, 1323–1329 (2010).

    CAS  Article  Google Scholar 

  15. Meyer, J.G. et al. Expanding proteome coverage with orthogonal-specificity α-lytic proteases. Mol. Cell. Proteomics 13, 823–835 (2014).

    CAS  Article  Google Scholar 

  16. López-Ferrer, D. et al. Pressurized pepsin digestion in proteomics: an automatable alternative to trypsin for integrated top-down bottom-up proteomics. Mol. Cell. Proteomics 10, M110.001479 (2011).

    Article  Google Scholar 

  17. Bian, Y. et al. Improve the coverage for the analysis of phosphoproteome of HeLa cells by a tandem digestion approach. J. Proteome Res. 11, 2828–2837 (2012).

    CAS  Article  Google Scholar 

  18. Choudhary, G., Wu, S.-L., Shieh, P. & Hancock, W.S. Multiple enzymatic digestion for enhanced sequence coverage of proteins in complex proteomic mixtures using capillary LC with ion trap MS/MS. J. Proteome Res. 2, 59–67 (2003).

    CAS  Article  Google Scholar 

  19. Huesgen, P.F. et al. LysargiNase mirrors trypsin for protein C-terminal and methylation-site identification. Nat. Methods 12, 55–58 (2015).

    CAS  Article  Google Scholar 

  20. Peng, M. et al. Protease bias in absolute protein quantitation. Nat. Methods 9, 524–525 (2012).

    CAS  Article  Google Scholar 

  21. Aye, T.T. et al. Proteome-wide protein concentrations in the human heart. Mol. Biosyst. 6, 1917–1927 (2010).

    CAS  Article  Google Scholar 

  22. Benevento, M. et al. Adenovirus composition, proteolysis, and disassembly studied by in-depth qualitative and quantitative proteomics. J. Biol. Chem. 289, 11421–11430 (2014).

    CAS  Article  Google Scholar 

  23. Low, T.Y. et al. Quantitative and qualitative proteome characteristics extracted from in-depth integrated genomics and proteomics analysis. Cell Rep. 5, 1469–1478 (2013).

    CAS  Article  Google Scholar 

  24. Gauci, S. et al. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. Anal. Chem. 81, 4493–4501 (2009).

    CAS  Article  Google Scholar 

  25. Mohammed, S. et al. Multiplexed proteomics mapping of yeast RNA polymerase II and III allows near-complete sequence coverage and reveals several novel phosphorylation sites. Anal. Chem. 80, 3584–3592 (2008).

    CAS  Article  Google Scholar 

  26. Richards, A.L. et al. One-hour proteome analysis in yeast. Nat. Protoc. 10, 701–714 (2015).

    CAS  Article  Google Scholar 

  27. Washburn, M.P., Wolters, D. & Yates, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247 (2001).

    CAS  Article  Google Scholar 

  28. Klammer, A.A. & MacCoss, M.J. Effects of modified digestion schemes on the identification of proteins from complex mixtures. J. Proteome Res. 5, 695–700 (2006).

    CAS  Article  Google Scholar 

  29. Wu, X., Xiong, E., Wang, W., Scali, M. & Cresti, M. Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis. Nat. Protoc. 9, 362–374 (2014).

    CAS  Article  Google Scholar 

  30. Schuchard, M.D. et al. Artifactual isoform profile modification following treatment of human plasma or serum with protease inhibitor, monitored by 2-dimensional electrophoresis and mass spectrometry. Biotechniques 39, 239–247 (2005).

    CAS  Article  Google Scholar 

  31. Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    CAS  Article  Google Scholar 

  32. Keller, B.O., Sui, J., Young, A.B. & Whittal, R.M. Interferences and contaminants encountered in modern mass spectrometry. Anal. Chim. Acta 627, 71–81 (2008).

    CAS  Article  Google Scholar 

  33. Good, D.M., Wirtala, M., McAlister, G.C. & Coon, J.J. Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteomics 6, 1942–1951 (2007).

    CAS  Article  Google Scholar 

  34. Molina, H., Horn, D.M., Tang, N., Mathivanan, S. & Pandey, A. Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 104, 2199–2204 (2007).

    CAS  Article  Google Scholar 

  35. Gershon, P.D. Cleaved and missed sites for trypsin, lys-C, and lys-N can be predicted with high confidence on the basis of sequence context. J. Proteome Res. 13, 702–709 (2014).

    CAS  Article  Google Scholar 

  36. Giansanti, P. et al. An augmented multiple-protease-based human phosphopeptide atlas. Cell Rep. 11, 1834–43 (2015).

    CAS  Article  Google Scholar 

  37. Boja, E.S. & Fales, H.M. Overalkylation of a protein digest with iodoacetamide. Anal. Chem. 73, 3576–3582 (2001).

    CAS  Article  Google Scholar 

  38. Meiring, H.D., van der Heeft, E., ten Hove, G.J. & de Jong, A.P.J.M. Nanoscale LC-MS(n): technical design and applications to peptide and protein analysis. J. Sep. Sci. 25, 557–568 (2002).

    CAS  Article  Google Scholar 

  39. Udeshi, N.D., Mertins, P., Svinkina, T. & Carr, S.A. Large-scale identification of ubiquitination sites by mass spectrometry. Nat. Protoc. 8, 1950–1960 (2013).

    CAS  Article  Google Scholar 

  40. Villén, J., Gygi, S.P. & Villen, J. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat. Protoc. 3, 1630–1638 (2008).

    Article  Google Scholar 

  41. Hohmann, L. et al. Proteomic analyses using Grifola frondosa metalloendoprotease Lys-N. J. Proteome Res. 8, 1415–1422 (2009).

    CAS  Article  Google Scholar 

  42. Taouatas, N., Heck, A.J.R. & Mohammed, S. Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions. J. Proteome Res. 9, 4282–4288 (2010).

    CAS  Article  Google Scholar 

  43. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    CAS  Article  Google Scholar 

  44. Wis´niewski, J.R. et al. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–352 (2009).

    Article  Google Scholar 

  45. Mallick, P. et al. Computational prediction of proteotypic peptides for quantitative proteomics. Nat. Biotechnol. 25, 125–131 (2007).

    CAS  Article  Google Scholar 

  46. Colaert, N., Helsens, K., Martens, L., Vandekerckhove, J. & Gevaert, K. Improved visualization of protein consensus sequences by iceLogo. Nat. Methods 6, 786–787 (2009).

    CAS  Article  Google Scholar 

  47. Köcher, T., Pichler, P., Swart, R. & Mechtler, K. Quality control in LC-MS/MS. Proteomics 11, 1026–1030 (2011).

    Article  Google Scholar 

  48. Köcher, T., Pichler, P., Swart, R. & Mechtler, K. Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients. Nat. Protoc. 7, 882–890 (2012).

    Article  Google Scholar 

Download references

Acknowledgements

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).

Author information

Authors and Affiliations

Authors

Contributions

A.J.R.H. conceived the idea for this protocol. P.G. and L.T. designed and performed the experiments and analyzed the data. All authors wrote the manuscript and discussed the experimental results.

Corresponding author

Correspondence to Albert J R Heck.

Ethics declarations

Competing interests

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 information

Supplementary Text and Figures

Supplementary Figure 1 (PDF 303 kb)

Supplementary Table 1

Recommended digestion conditions, availability and purity of the here used proteases. (XLSX 14 kb)

Supplementary Table 2

Typical elution profile of tryptic BSA peptides (20 fmole injection) during a 45min chromatographic gradient. (XLSX 9 kb)

Supplementary Table 3

List of BSA peptides identified in each proteolytic digest and their benchmark against theoretical digestion. (XLSX 52 kb)

Supplementary Table 4

List of E. coli proteins and peptides identifications in each proteolytic digest using the enzyme-specific search settings. (XLSX 46899 kb)

Supplementary Table 5

List of E. coli proteins and peptides identifications in each proteolytic digest using the non-specific search settings. (XLSX 58541 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.057

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing