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Membrane proteomics: ‘sweet dreams’ reviving
Author(s) -
Ghosh Dhimankrishna,
Cheng Keding,
Beavis Ronald,
Wilkins John Alfred
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a927-a
Subject(s) - membrane protein , lectin , trypsin , biochemistry , glycoprotein , chemistry , transmembrane protein , membrane , proteomics , affinity chromatography , membrane glycoproteins , enzyme , receptor , gene
Despite several shortcomings, membrane proteins continue to represent 2/3 rd of new biopharmaceuticals’ targets. It is likely that further improvements in the global expression and distribution of membrane proteins will expedite the development of future drug targets. In our present study we have re‐evaluated the possibility of exploring lectin affinity as an alternate method to study the distribution of membrane proteins in comparison to membrane isolation method. Methodology Isolated membrane from an erythroleukaemic cell line (K562) was washed with the reagents, known to remove non‐covalently associated proteins from the membrane. The proteins of these ‘stripped’ membranes were separated by SDS‐PAGE, in gel digested with trypsin and analyzed by nano‐LC‐ESI‐MS/MS. In a separate experiment cellular glycoproteins were isolated by lectin affinity chromatography employing Con A and WGA lectins. Isolated glycoproteins were digested in‐solution with trypsin and peptide fragments were analyzed by an off‐line RP‐HPLC and MALDI‐TOF. Protein identification was performed using the GPM algorithm. Results A total 624 proteins were identified from the isolated microsome. Of these proteins, 33% were predicted as transmembrane(TM) proteins. Approximately 10% of these transmembrane proteins were also isolated by lectin approach while 9% of lectin purified TM proteins were not identified in microsomal preparation. Conclusion Membrane proteins isolated from two different procedures seem to be complementary to each other. In future, these two procedures may be performed in tandem to increase the representation of membrane proteins in the preparation.