Post-digestion ¹⁸O Exchange/Labeling for Quantitative Shotgun Proteomics of Membrane Proteins

Abstract

The role of membrane proteins is critical for regulation of physiologic and pathologic cellular processes. Hence it is not surpassing that membrane proteins make ∼70% of contemporary drug targets. Quantitative profiling of membrane proteins using mass spectrometry (MS)-based proteomics is critical in a quest for disease biomarkers and novel cancer drugs. Post-digestion ¹⁸O exchange is a simple and efficient method for differential ¹⁸O/¹⁶O stable isotope labeling of two biologically distinct specimens, allowing relative quantitation of proteins in complex mixtures when coupled with shotgun MS-based proteomics. Due to minimal sample consumption and unrestricted peptide tagging, ¹⁸O/¹⁶O stable isotope labeling is particularly suitable for amount-limited protein specimens typically encountered in membrane and clinical proteomics. This chapter describes a protocol that relies on shotgun proteomics for quantitative profiling of the detergent-insoluble membrane proteins isolated from HeLa cells, differentially transfected with plasmids expressing HIV Gag protein and its myristylation-defective N-terminal mutant. Whilst this protocol depicts solubilization of detergent-insoluble membrane proteins coupled with post-digestion ¹⁸O labeling, it is amenable to any complex membrane protein mixture. Described approach relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (v/v) methanol followed by differential ¹⁸O/¹⁶O labeling of protein digests in 20% (v/v) methanol buffer. After mixing, the differentially labeled peptides are fractionated using off-line strong cation exchange (SCX) followed by on-line reversed phase nanoflow reversed-phase liquid chromatography (nanoRPLC)-MS identification/quantiation of peptides/proteins. The use of methanol-based buffers in the context of the post-digestion ¹⁸O exchange/labeling eliminates the need for detergents or chaotropes that interfere with LC separations and peptide ionization. Sample losses are minimized because solubilization, digestion, and stable isotope labeling are carried out in a single tube, avoiding any sample transfer or buffer exchange between these steps.