Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing.
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.
Seebacher
Jan
J
Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
Mallick
Parag
P
Zhang
Ning
N
Eddes
James S
JS
Aebersold
Ruedi
R
Gelb
Michael H
MH
D002853:Chromatography, Liquid; D019295:Computational Biology / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003432:Cross-Linking Reagents; D011506:Proteins