Trypsin-mediated ¹⁸O/¹⁶O labeling for biomarker discovery.

Differential (18)O/(16)O stable isotopic labeling that relies on post-digestion (18)O exchange is a simple and efficient method for the relative quantitation of proteins in complex mixtures. This method incorporates two (18)O atoms onto the C-termini of proteolytic peptides resulting in a 4 Da mass-tag difference between (18)O- and (16)O-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 (18)O 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 (18)O 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 (18)O/(16)O 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 (18)O 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 (18)O/(16)O 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.