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Separation of human erythrocyte membrane associated proteins with one‐dimensional and two‐dimensional gel electrophoresis followed by identification with matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry
Author(s) -
Low Teck Yew,
Seow Teck Keong,
Chung Maxey C. M.
Publication year - 2002
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200209)2:9<1229::aid-prot1229>3.0.co;2-n
Subject(s) - gel electrophoresis , chemistry , two dimensional gel electrophoresis , proteome , mass spectrometry , proteomics , polyacrylamide gel electrophoresis , difference gel electrophoresis , chromatography , trypsinization , membrane protein , biochemistry , molecular weight size marker , molecular mass , microbiology and biotechnology , trypsin , biology , membrane , enzyme , gene
Abstract A classical proteomic analysis was used to establish a reference map of proteins associated with healthy human erythrocyte ghosts. Following osmotic lysis and differential centrifugation, ghost proteins were separated by either one‐dimensional gel electrophoresis (1‐DE) or two‐dimensional gel electrophoresis (2‐DE). Selected protein bands or spots were excised and trypsinized before mass spectrometric analyses and data mining was performed using the SWISS‐PROT and NCBI nonredundant databases. A total of 102 protein spots from a 2‐D gel were successfully identified. These corresponded to 59 distinct polypeptides with the remaining 43 being isoforms. As for the 1‐D gel, 44 polypeptides were identified, of which 19 were also found on the 2‐D gel. Most of the 19 common polypeptides were membrane cytoskeletal proteins that are often referred to as the “band” proteins. The remaining 25 polypeptides that were found exclusively on 1‐D gels were proteins with high hydrophobicity ( e.g. , sorbitol dehydrogenase and glucose transporter) and high molecular mass ( e.g. , Kell blood group glycoprotein and Janus‐kinase 2). A higher number of signaling proteins was also identified on 1‐D gels compared to 2‐D gels. These included Ras, cAMP dependent protein kinase and TGF‐beta receptor type 1 precursor.