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Trypsin catalyzed 16O-to-18O exchange for comparative proteomics: Tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers

  • Manfred HellerEmail author
  • Hassan Mattou
  • Christoph Menzel
  • Xudong Yao
Focus: Proteomics

Abstract

Quantitative or comparative proteome analysis was initially performed with 2-dimensional gel electrophoresis with the inherent disadvantages of being biased towards certain proteins and being labor intensive. Alternative mass spectrometry-based approaches in conjunction with gel-free protein/peptide separation have been developed in recent years using various stable isotope labeling techniques. Common to all these techniques is the incorporation, biosynthetically or chemically, of a labeling moiety having either a natural isotope distribution of hydrogen, carbon, oxygen, or nitrogen (light form) or being enriched with heavy isotopes like deuterium, 13C, 18O, or 15N, respectively. By mixing equal amounts of a control sample possessing for instance the light form of the label with a heavy-labeled case sample, differentially labeled peptides are detected by mass spectrometric methods and their intensities serve as a means for direct relative protein quantification. While each of the different labeling methods has its advantages and disadvantages, the endoprotease 16O-to-18O catalyzed oxygen exchange at the C-terminal carboxylic acid is extremely promising because of the specificity assured by the enzymatic reaction and the labeling of essentially every protease-derived peptide. We show here that this methodology is applicable to complex biological samples such as a subfraction of human plasma. Furthermore, despite the relatively small mass difference of 4 Da between the two labeled forms, corresponding to the exchange of two oxygen atoms by two 18O isotopes, it is possible to quantify differentially labeled proteins on an ion trap mass spectrometer with a mass resolution of about 2000 in automated data dependent LC-MS/MS acquisition mode. Post column sample deposition on a MALDI target parallel to on-line ESI-MS/MS enables the analysis of the same compounds by means of ESI- and MALDI-MS/MS. This has the potential to increase the confidence in the quantification results as well as to increase the sequence coverage of potentially interesting proteins by complementary peptide ionization techniques. Additionally the paired y-ion signals in tandem mass spectra of 16O/18O-labeled peptide pairs provide a means to confirm automatic protein identification results or even to assist de novo sequencing of yet unknown proteins.

Keywords

Isotopic Peak Peptide Pair Oxygen Exchange Reaction Relative Protein Quantification Natural Isotope Distribution 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© American Society for Mass Spectrometry 2003

Authors and Affiliations

  • Manfred Heller
    • 1
    Email author
  • Hassan Mattou
    • 1
  • Christoph Menzel
    • 1
  • Xudong Yao
    • 1
  1. 1.GeneProt Inc.Meyrin 2Switzerland

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