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FEBS J. 2015 Jul;282(14):2612-26. doi: 10.1111/febs.13287. Epub 2015 Apr 14.

Proteomics beyond trypsin.

Author information

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

Abstract

Peptide-centered shotgun analysis of proteins has been the core technology in mass spectrometry based proteomics and has enabled numerous biological discoveries, such as the large-scale charting of protein-protein interaction networks, the quantitative analysis of protein post-translational modifications and even the first drafts of the human proteome. The conversion of proteins into peptides in these so-called bottom-up approaches is nearly uniquely done by using trypsin as a proteolytic reagent. Here, we argue that our view of the proteome still remains incomplete and this is partially due to the nearly exclusive use of trypsin. Newly emerging alternative proteases and/or multi-protease protein digestion aim to increase proteome sequence coverage and improve the identification of post-translational modifications, through the analysis of complementary and often longer peptides, introducing an approach termed middle-down proteomics. Of pivotal importance for this purpose is the identification of proteases beneficial for use in proteomics. Here, we describe some of the shortcomings of the nearly exclusive use of trypsin in proteomics and review the properties of other proteomics-appropriate proteases. We describe favorable protease traits with an emphasis on middle-down proteomics and suggest potential sources for the discovery of new proteases. We also highlight a few examples wherein the use of other proteases than trypsin enabled the generation of more comprehensive data sets leading to previously unexplored knowledge of the proteome.

KEYWORDS:

bias in quantitative proteomics; cleavage specificity; digestion; mass spectrometry; middle-down proteomics; proteases; protein post-translational modifications; shotgun proteomics

PMID:
25823410
DOI:
10.1111/febs.13287
[Indexed for MEDLINE]
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