Protocol | Published:

Six alternative proteases for mass spectrometry–based proteomics beyond trypsin

Nature Protocols volume 11, pages 9931006 (2016) | Download Citation

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.

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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

Author notes

    • Piero Giansanti
    •  & Liana Tsiatsiani

    These authors contributed equally to this work.

Affiliations

  1. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands.

    • Piero Giansanti
    • , Liana Tsiatsiani
    • , Teck Yew Low
    •  & Albert J R Heck
  2. Netherlands Proteomics Centre, Utrecht, the Netherlands.

    • Piero Giansanti
    • , Liana Tsiatsiani
    • , Teck Yew Low
    •  & Albert J R Heck

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Albert J R Heck.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figure 1

Excel files

  1. 1.

    Supplementary Table 1

    Recommended digestion conditions, availability and purity of the here used proteases.

  2. 2.

    Supplementary Table 2

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

  3. 3.

    Supplementary Table 3

    List of BSA peptides identified in each proteolytic digest and their benchmark against theoretical digestion.

  4. 4.

    Supplementary Table 4

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

  5. 5.

    Supplementary Table 5

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

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DOI

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

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