title: Quantitative proteomics analysis of high-density lipoproteins by stable 18O-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 affinity 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 specific considerations for this particle, paving the way to the in-depth study of the protein cargo of HDL and its implication in cardiovascular diseases.
authors.affiliation: Vascular Research Lab IIS-Fundación Jiménez Díaz, Instituto de Investigacion Sanitaria Fundacion Jimenez Diaz (IIS-FJD), Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Burillo
authors.forename: Jesus
authors.initials: J
authors.lastname: Vazquez
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Jorge
mesh_terms: D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007526:Isoelectric Point; D007553:Isotope Labeling; D008075:Lipoproteins, HDL / Q000097:blood / Q000737:chemistry* / Q000378:metabolism*; D010103:Oxygen Isotopes / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D008075:Lipoproteins, HDL; D010103:Oxygen Isotopes; D010446:Peptide Fragments