Rationale: Trypsin is an important digestive enzyme in peptide sample preparation for proteomics. It digests proteins at the C-terminal of Arg or Lys residues. The majority of commercial products are obtained from animal sources. In a previous study, we reported the production process for recombinant trypsin (r-trypsin) and acetylated trypsin (r-Ac-trypsin). In this paper, we want to evaluate whether the r-trypsin and r-Ac-trypsin are suitable for proteomics research.

Methods: The trypsins used in this research were first normalized to the same concentration and used for further evaluation. The stability and buffer compatibility (2M urea, 0.1% SDS and 10% acetonitrile) were compared and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The digestion efficiency and specificity were compared based on a simple protein substrate, human serum albumin (HSA) and a complex proteomic sample, yeast lysate. The acquisition of proteomics data was achieved by ultra-high performance liquid chromatography (UPLC) connected to an LTQ Orbitrap Velos mass spectrometer.

Results: r-Ac-trypsin demonstrated similar tolerance to 2 M urea and 10% acetonitrile but weaker 0.1% SDS tolerance than commercial trypsins. Based on simple protein sample HSA, the activity and specificity of r-Ac-trypsin were similar to that of commercial trypsins. However, it demonstrated superior activity and specificity on complicated samples like yeast lysate. More interestingly, the newly developed r-Ac-trypsin was more resistant to autolysis, which enabled more complete digestion of proteomic samples.

Conclusions: The r-Ac-trypsin described here is a recombinant product. In addition it showed similar or superior properties such as stability activity and specificity to commercial products. It can be used in peptide sample preparation in proteomics studies.

• PMID: 27003043
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Reference Type

Journal Article | Research Support, Non-U.S. Gov't

Authors

Wu F, Zhao M, Zhang Y, Su N, Xiong Z, Xu P

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