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Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization
Published on: February 27, 2020
Recombinant acetylated trypsin demonstrates superior stability and higher activity than commercial products in
Feilin Wu1,2, Mingzhi Zhao2, Yao Zhang2,3
1Life Science College, Southwest Forestry University, Kunming, 650224, P.R. China.
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.
