Quantitative proteomics analysis of high-density lipoproteins by stable 18 O-Isotope labeling

Abstract

For the large-scale study of dynamic proteomes, quantitative proteomic approaches based on stable isotope labeling and mass spectrometry (MS) have been developed as a high-throughput, reproducible, and robust alternative to conventional gel-based techniques. In this chapter, we describe in detail a quantitative proteomic strategy based on HDL isolation by af fi nity chromatography, in-gel trypsin digestion of protein extracts, peptide 18 O labeling, separation by off-gel isoelectric focusing, and peptide analysis on a linear ion trap mass spectrometer, followed by the application of a robust multilayered statistical model. This protocol is of universal applicability and has been successfully applied to the global characterization of the HDL proteome with some speci fi c considerations for this particle, paving the way to the in-depth study of the protein cargo of HDL and its implication in cardiovascular diseases. © 2013 Springer Science+Business Media New York.