Trypsin-Mediated 18O/16O Labeling for Biomarker Discovery
Differential 18O/16O stable isotopic labeling that relies on post-digestion 18O exchange is a simple and efficient method for the relative quantitation of proteins in complex mixtures. This method incorporates two 18O atoms onto the C-termini of proteolytic peptides resulting in a 4 Da mass-tag difference between 18O- and 16O-labeled peptides. This allows for wide-range relative quantitation of proteins in complex mixtures using shotgun proteomics. Because of minimal sample consumption and unrestricted peptide tagging, the post-digestion 18O exchange is suitable for labeling of low-abundance membrane proteins enriched from cancer cell lines or clinical specimens, including tissues and body fluids. This chapter describes a protocol that applies post-digestion 18O labeling to elucidate putative endogenous tumor hypoxia markers in the plasma membrane fraction enriched from a hypoxia-adapted malignant melanoma cell line. Plasma membrane proteins from hypoxic and normoxic cells were differentially tagged using 18O/16O stable isotopic labeling. The initial tryptic digestion and solubilization of membrane proteins were carried out in a buffer containing 60 % methanol followed by post-digestion 18O exchange/labeling in buffered 20 % methanol. The differentially labeled peptides were mixed in a 1:1 ratio and fractionated using off-line strong cation exchange (SCX) liquid chromatography followed by on-line reversed-phase nano-flow RPLC-MS identification and quantitation of peptides/proteins in respective SCX fractions. The present protocol illustrates the utility of 18O/16O stable isotope labeling in the context of quantitative shotgun proteomics that provides a basis for the discovery of hypoxia-induced membrane protein markers in malignant melanoma cell lines.
Key words18O/16O stable isotope labeling Hypoxia biomarker Melanoma Quantitative shotgun proteomics Mass spectrometry
This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contracts HHSN261200800001E and NO1-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the United States Government.