A proteomic characterization of the plasma membrane of human epidermis by high-throughput mass spectrometry.
Membrane proteins are responsible for many critical cellular functions and identifying cell surface proteins on different keratinocyte populations by proteomic approaches would improve our understanding of their biological function. The ability to characterize membrane proteins, however, has lagged behind that of soluble proteins both in terms of throughput and protein coverage. In this study, a membrane proteomic investigation of keratinocytes using a two-dimensional liquid chromatography (LC) tandem-mass spectrometry (MS/MS) approach that relies on a buffered methanol-based solubilization, and tryptic digestion of purified plasma membrane is described. A highly enriched plasma membrane fraction was prepared from newborn foreskins using sucrose gradient centrifugation, followed by a single-tube solubilization and tryptic digestion of membrane proteins. This digestate was fractionated by strong cation-exchange chromatography and analyzed using microcapillary reversed-phase LC-MS/MS. In a set of 1306 identified proteins, 866 had a gene ontology (GO) annotation for cellular component, and 496 of these annotated proteins (57.3%) were assigned as known integral membrane proteins or membrane-associated proteins. Included in the identification of a large number of aqueous insoluble integral membrane proteins were many known intercellular adhesion proteins and gap junction proteins. Furthermore, 121 proteins from cholesterol-rich plasma membrane domains (caveolar and lipid rafts) were identified.
Blonder
Josip
J
Laboratory of Proteomics and Analytical Technologies, Mass Spectrometry Center, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland, USA.
Terunuma
Atsushi
A
Conrads
Thomas P
TP
Chan
King C
KC
Yee
Carole
C
Lucas
David A
DA
Schaefer
Carl F
CF
Yu
Li-Rong
LR
Issaq
Haleem J
HJ
Veenstra
Timothy D
TD
Vogel
Jonathan C
JC
D021941:Caveolae / Q000737:chemistry; D015815:Cell Adhesion Molecules / Q000032:analysis; D002462:Cell Membrane / Q000737:chemistry; D000078404:Epidermal Cells; D004817:Epidermis / Q000737:chemistry*; D006801:Humans; D007231:Infant, Newborn; D007365:Intercellular Junctions / Q000737:chemistry; D015603:Keratinocytes / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D021962:Membrane Microdomains / Q000737:chemistry; D040901:Proteomics
D015815:Cell Adhesion Molecules