Pharmaceutical Sciences Faculty Publications
A Proteomic Characterization of the Plasma Membrane of Human Epidermis by High-Throughput Mass Spectrometry
Document Type
Article
Publication Date
10-1-2004
Journal Title
The Journal of Investigative Dermatology
ISSN
0022-202X
Volume
123
Issue
4
First Page
691
Last Page
699
DOI
10.1111/j.0022-202X.2004.23421.x
PubMed ID
15373774
Abstract
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.
Keywords
Caveolae, cell adhesion molecules, cell membrane, epidermal cells, epidermis, intercellular junctions, keratinocytes, mass spectrometry, membrane microdomains, proteomics
Recommended Citation
Blonder, Josip; Terunuma, Atsushi; Conrads, Thomas P.; Chan, King C.; Yee, Carole; Lucas, David A.; Schaefer, Carl F.; Yu, Li-Rong; Issaq, Haleem J.; Veenstra, Timothy D.; and Vogel, Jonathan C., "A Proteomic Characterization of the Plasma Membrane of Human Epidermis by High-Throughput Mass Spectrometry" (2004). Pharmaceutical Sciences Faculty Publications. 458.
https://digitalcommons.cedarville.edu/pharmaceutical_sciences_publications/458