title: Identification of components of protein complexes using a fluorescent photo-cross-linker and mass spectrometry.
abstract: This study describes a novel method for improving the specific recognition, detection, and identification of proteins involved in multiprotein complexes. The method is based on a combination of coimmunoprecipitation, chemical cross-linking, and specific fluorescent tagging of protein components in close association with one another. Specific fluorescent tagging of the protein complex components was achieved using the cleavable, fluorescent cross-linker sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido) ethyl-1,3'-dithiopropionate (SAED). Following dissociation and separation by SDS-PAGE, the fluorescently tagged proteins are then visualized by UV illumination, excised, and, following in-gel digestion, identified by mass spectrometry. In this study, a complex of the HIV-envelope protein gp120 and its cellular receptor CD4 was used as a model system. The sensitivity of detection of fluorescent SAED-labeled proteins in SDS gels, and the sensitivity of the mass spectrometric identification of fluorescent proteins after in-gel digestion, is in the range of a few hundred femtomoles of protein. This sensitivity is comparable to that achieved with silver-staining techniques, but fluorescence detection is protein independent and no background interference occurs. Furthermore, fluorescence labeling is significantly more compatible with mass spectrometric identification of proteins than is silver staining. The first application of this strategy was in the investigation of the mechanism of spermiation, the process by which mature spermatids separate from Sertoli cells. For the coimmunoprecipitation experiment, an antibody against paxillin, a protein involved in spermatid-Sertoli cell junctional complexes, was used. More components of the paxillin protein complex were visible by fluorescence detection of SAED-labeled proteins than were visible on comparable silver-stained gels. Mass spectrometric analysis of the fluorescently labeled proteins identified integrin alpha6 precursor as a protein associated in a complex with paxillin. The identification of integrin alpha6 precursor was confirmed by Western blot analysis and verifies the applicability of this novel approach for identifying proteins involved in protein complexes.
authors.affiliation: Laboratory of Toxicology, National Institute of Environmental Health Sciences/NIH, Research Triangle Park, North Carolina 27713, USA.
authors.forename: Robert N
authors.initials: RN
authors.lastname: Wine
authors.forename: John M
authors.initials: JM
authors.lastname: Dial
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Tomer
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
mesh_terms: D000818:Animals; D015704:CD4 Antigens / Q000032:analysis / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000378:metabolism; D005456:Fluorescent Dyes; D015699:HIV Envelope Protein gp120 / Q000032:analysis / Q000378:metabolism; D006801:Humans; D039503:Integrin alpha6 / Q000032:analysis / Q000378:metabolism; D007365:Intercellular Junctions / Q000737:chemistry; D046911:Macromolecular Substances; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D046912:Multiprotein Complexes; D051419:Paxillin; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D010777:Photochemistry; D011485:Protein Binding; D011506:Proteins / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013087:Spermatids / Q000737:chemistry
chemical_list: D015704:CD4 Antigens; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D005456:Fluorescent Dyes; D015699:HIV Envelope Protein gp120; D039503:Integrin alpha6; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011506:Proteins; C496063:Pxn protein, rat