title: Recombinant acetylated trypsin demonstrates superior stability and higher activity than commercial products in quantitative proteomics studies.
abstract: BACKGROUND
  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.
authors.affiliation: Life Science College, Southwest Forestry University, Kunming, 650224, P.R. China.|State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing, 102206, P.R. China.
authors.forename: Feilin
authors.initials: F
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing, 102206, P.R. China.
authors.forename: Mingzhi
authors.initials: M
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing, 102206, P.R. China.|Institute of Microbiology, Chinese Academy of Science, Beijing, 100101, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing, 102206, P.R. China.
authors.forename: Na
authors.initials: N
authors.lastname: Su
authors.affiliation: Life Science College, Southwest Forestry University, Kunming, 650224, P.R. China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Xiong
authors.affiliation: State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing, 102206, P.R. China.|Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan, 430071, P.R. China.|Anhui Medical University, Hefei, 230032, Anhui, P.R. China.
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
mesh_terms: D004795:Enzyme Stability; D013058:Mass Spectrometry; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014357:Trypsin