Analysis of proteins copurifying with the CD4/lck complex using one-dimensional polyacrylamide gel electrophoresis and mass spectrometry: comparison with affinity-tag based protein detection and evaluation of different solubilization methods.
Mass spectrometry-based identification of the components of affinity purified protein complexes after polyacrylamide gel electrophoresis (PAGE) and in-gel digest has become very popular for the detection of novel protein interactions. As an alternative, the entire protein complex can be subjected to proteolytic cleavage followed by chromatographic separation of the peptides. Based on our earlier report of a method using affinity tag-mediated purification of cysteine-containing peptides to analyse proteins present in an affinity purification of the CD4/lck receptor complex, we here evaluated the use of one-dimensional polyacrylamide gel electrophoresis for analysis of the same receptor complex purification. Using electrospray and tandem mass spectrometry analyses of tryptic peptides from in-gel digested proteins we identified the components of the CD4 receptor complex along with 23 other proteins that were all likely to be non-specifically binding proteins and mainly different from the proteins detected in our previous study. We compare the alternative strategy with the affinity tag-based method that we described earlier and show that the PAGE-based method enables more proteins to be identified. We also evaluated the use of a more stringent lysis buffer for the CD4 purification to minimise non-specific binding and identified 52 proteins along with CD4 in three independent experiments suggesting that the choice of lysis buffer had no significant effect on the extent of non-specific binding. Non-specific binding was inconsistent and involved various types of proteins underlining the importance of reproducibility and control experiments in proteomic studies.
Bernhard
Oliver K
OK
Center for Virus Research, Westmead Millennium Institute, National Center for HIV Virology Research, Westmead Hospital and the University of Sydney, Westmead, New South Wales, Australia.
Cunningham
Anthony L
AL
Sheil
Margaret M
MM
D000595:Amino Acid Sequence; D002021:Buffers; D015704:CD4 Antigens / Q000378:metabolism*; D002460:Cell Line; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D046911:Macromolecular Substances; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D012394:Rosaniline Dyes; D012995:Solubility
D002021:Buffers; D015704:CD4 Antigens; D003902:Detergents; D046911:Macromolecular Substances; D011506:Proteins; D012394:Rosaniline Dyes; C048139:Coomassie blue; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)