Incorporation of ¹⁸Oxygen into Peptide Mixtures and Analysis with Multi‐Dimensional Chromatography and Mass‐Spectroscopy

Abstract

Enzymatic labeling with ¹⁸oxygen has the potential to become a widely applied method of isotope labeling for differential protein expression analysis by mass spectrometry because it is not amino acid specific and the reagents are cost‐effective and readily available. In this work, we investigate experimental parameters that affect efficient ¹⁸O incorporation with a model bovine serum albumin protein system and then use optimized chemistries for labeling the c‐terminus of peptides in a yeast proteome. Additionally, the role of sample handling, including the use of liquid chromatography was examined. An analytical methodology was developed which demonstrates the application of multi‐dimensional chromatography in conjunction with enzymatic labeling.

Keywords:

• Proteomics
• mass spectrometry
• LC‐MS
• ¹⁸O labeling
• isotope
• peptide
• chromatography
• Saccharomyces cerevisiae

Acknowledgments

The authors thank J. Heitman for the gift of the MLY61 yeast strains. A. Rahbar is and I. Medintz was a National Research Council Postdoctoral Fellow. M. Lassman was an American Society of Electrical Engineering Postdoctoral Fellow. The research was supported by NRL work unit 6027 and the internal CBMSE Systems Biology Initiative, G. Vora P.I.