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A Simple Carbamidomethylation-Based Isotope Labeling Method for Quantitative Shotgun Proteomics

  • Oh, Donggeun (Department of Bio-Analytical Science, University of Science & Technology) ;
  • Lee, Sun Young (Center for Bioanalysis, Division of Metrology for Quality of Life, Korea Research Institute of Standards and Science) ;
  • Kwon, Meehyang (Center for Bioanalysis, Division of Metrology for Quality of Life, Korea Research Institute of Standards and Science) ;
  • Kim, Sook-Kyung (Center for Bioanalysis, Division of Metrology for Quality of Life, Korea Research Institute of Standards and Science) ;
  • Moon, Myeong Hee (Department of Chemistry, Yonsei University) ;
  • Kang, Dukjin (Center for Bioanalysis, Division of Metrology for Quality of Life, Korea Research Institute of Standards and Science)
  • Received : 2014.06.25
  • Accepted : 2014.07.01
  • Published : 2014.09.30

Abstract

In this study, we present a new isotope-coded carbamidomethylation (iCCM)-based quantitative proteomics, as a complementary strategy for conventional isotope labeling strategies, with providing the simplicity, ease of use, and robustness. In iCCM-based quantification, two proteome samples can be separately isotope-labeled by means of covalently reaction of all cysteinyl residues in proteins with iodoacetamide (IAA) and its isotope (IAA-$^{13}C_2$, $D_2$), denoted as CM and iCCM, respectively, leading to a mass shift of all cysteinyl residues to be + 4 Da. To evaluate iCCM-based isotope labeling in proteomic quantification, 6 protein standards (i.e., bovine serum albumin, serotransferrin, lysozyme, beta-lactoglobulin, beta-galactosidase, and alpha-lactalbumin) isotopically labeled with IAA and its isotope, mixed equally, and followed by proteolytic digestion. The resulting CM-/iCCM-labeled peptide mixtures were analyzed using a nLC-ESI-FT orbitrap-MS/MS. From our experimental results, we found that the efficiency of iCCM-based quantification is more superior to that of mTRAQ, as a conventional nonisobaric labeling method, in which both of a number of identified peptides from 6 protein standards and the less quantitative variations in the relative abundance ratios of heavy-/light-labeled corresponding peptide pairs. Finally, we applied the developed iCCM-based quantitative method to lung cancer serum proteome in order to evaluate the potential in biomarker discovery study.

Keywords

References

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