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Protein Cross-Linking Analysis Using Mass Spectrometry, Isotope-Coded Cross-Linkers, and Integrated Computational Data Processing

2006; American Chemical Society; Volume: 5; Issue: 9 Linguagem: Inglês

10.1021/pr060154z

1535-3907

J. Seebacher, Parag Mallick, Ning Zhang, James S. Eddes, Ruedi Aebersold, Michael H. Gelb,

Advanced Proteomics Techniques and Applications

Distance constraints in proteins and protein complexes provide invaluable information for calculation of 3D structures, identification of protein binding partners and localization of protein−protein contact sites. We have developed an integrative approach to identify and characterize such sites through the analysis of proteolytic products derived from proteins chemically cross-linked by isotopically coded cross-linkers using LC-MALDI tandem mass spectrometry and computer software. This method is specifically tailored toward the rapid analysis of low microgram amounts of proteins or multimeric protein complexes cross-linked with nonlabeled and deuterium-labeled bis-NHS ester cross-linking reagents (both commercially available and readily synthesized). Through labeling with [18O]water solvent and LC-MALDI analysis, the method further allows the possible distinction between Type 0 and Type 1 or Type 2 modified peptides (monolinks and looplinks or cross-links), although such a distinction is more readily made from analysis of tandem mass spectrometry data. When applied to the bacterial Colicin E7 DNAse/Im7 heterodimeric protein complex, 23 cross-links were identified including six intersubunit cross-links, all between residues that are close in space when examined in the context of the X-ray structure of the heterodimer. In addition, cross-links were successfully identified in five single subunit proteins, beta-lactoglobulin, cytochrome c, lysozyme, myoglobin, and ribonuclease A, establishing the generality of the approach. Keywords: protein structure • protein structure prediction • protein−protein interaction • cross-linking • mass spectrometry • proteomics • protein complex