Comparative proteomics of human endothelial cell caveolae and rafts using two-dimensional gel electrophoresis and mass spectrometry.
The human endothelial cell plasma membrane harbors two subdomains of similar lipid composition, caveolae and rafts, both crucially involved in various essential cellular processes like transcytosis, signal transduction and cholesterol homeostasis. Caveolin-enriched membranes, isolated by either cationic silica or buoyant density methods, were explored by comparing large series of two-dimensional (2-D) maps and subsequent identification of over 100 protein spots by matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprinting. Improved representation and identification of membrane proteins and valuable information on various post-translational modifications was achieved by the presented optimized procedures for solubilization, destaining and database searching/computing. Whereas the cationic silica purification yielded predominantly known endoplasmic reticulum residents, the cold-detergent method yielded a large number of known caveolae residents, including caveolin-1. Thus, a large part of this subproteome was established, including known (trans-)membrane, signal transduction and glycosyl phosphatidylinositol (GPI)-anchored proteins. Several predicted proteins from the human genome were isolated for the first time from biological samples, including SGRP58, SLP-2, C8ORF2, and XRP-2. These findings and various optimized procedures can serve as a reference to study the differential composition of endothelial cell caveolae and rafts, known to be involved in pathologies like cancer and cardiovascular disease.
Sprenger
Richard R
RR
Department of Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. a.j.horrevoets@amc.uva.nl
Speijer
Dave
D
Back
Jaap Willem
JW
De Koster
Chris G
CG
Pannekoek
Hans
H
Horrevoets
Anton J G
AJ
D001798:Blood Proteins / Q000032:analysis / Q000302:isolation & purification; D021941:Caveolae / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D042783:Endothelial Cells / Q000737:chemistry* / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000166:cytology*; D005136:Eye Proteins / Q000032:analysis / Q000302:isolation & purification; D019204:GTP-Binding Proteins; D015894:Genome, Human; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification*; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014471:Umbilical Veins
D001798:Blood Proteins; C121703:ERLIN2 protein, human; D005136:Eye Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; C072178:RP2 protein, human; C406287:STOML2 protein, human; D019204:GTP-Binding Proteins