Mass spectrometry analysis of CD4-associating proteins using affinity chromatography and affinity tag-mediated purification of tryptic peptides.
The study of protein interactions using mass spectrometry (MS) for identification of the components of purified protein complexes is leading to the description of increasingly valuable data on protein function. Commonly proteins in a given complex are identified via MS analysis of in-gel digests of gel electrophoretically separated proteins. In this study, we have evaluated the use of an approach employing the digest of the whole protein complex to identify directly the proteins present in a purification of the CD4 receptor complex. We used a cysteinyl affinity capture method to reduce the complexity of the peptide mixture that was obtained from the tryptic digest of the whole protein complex to the rather limited mixture of only cysteine-containing peptides. Here we report the use of this approach with MS for identification of the CD4 receptor complex components CD4 and p56lck, along with several other proteins present in the detergent-solubilized fractions from the purification. We have been able to identify these proteins using peptide sequence data obtained from cysteine-containing peptides. With appropriate control experiments, we have demonstrated the specific nature of the CD4-p56lck interaction. In contrast, the other proteins identified are shown to arise from nonspecific interactions during the affinity chromatography purification suggesting a possible loss of specific interactions during the chromatography procedure. We found that the complexity of the mixture was reduced such that only 10% of the peptides derived from tryptic digest of the identified proteins were detected. This represents only one-third of the cysteine-containing peptides, however, suggesting that this approach does not enable detection of all individual proteins.
Bernhard
Oliver K
OK
Centre for Virus Research, Westmead Milennium Institute, National Centre for HIV Virology Research, Westmead Hospital and The University of Sydney, Westmead NSW, Australia.
Burgess
Jennifer A
JA
Hochgrebe
Tim
T
Sheil
Margaret M
MM
Cunningham
Anthony L
AL
D001360:Avidin / Q000378:metabolism; D019921:Biotinylation; D015704:CD4 Antigens / Q000096:biosynthesis*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D003545:Cysteine / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D013053:Spectrophotometry; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology*
D015704:CD4 Antigens; D003902:Detergents; D010455:Peptides; D001360:Avidin; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D014357:Trypsin; D003545:Cysteine