title: Photo-assisted peptide enrichment in protein complex cross-linking analysis of a model homodimeric protein using mass spectrometry.
abstract: MS analysis of cross-linked peptides can be used to probe protein contact sites in macromolecular complexes. We have developed a photo-cleavable cross-linker that enhances peptide enrichment, improving the signal-to-noise ratio of the cross-linked peptides in mass spectrometry analysis. This cross-linker utilizes nitro-benzyl alcohol group that can be cleaved by UV irradiation and is stable during the multiple washing steps used for peptide enrichment. The enrichment method utilizes a cross-linker that aids in eliminating contamination resulting from protein-based retrieval systems, and thus, facilitates the identification of cross-linked peptides. Homodimeric pilM protein from Pseudomonas aeruginosa 2192 (pilM) was investigated to test the specificity and experimental conditions. As predicted, the known pair of lysine side chains within 14 Å was cross-linked. An unexpected cross-link involving the protein's amino terminus was also detected. This is consistent with the predicted mobility of the amino terminus that may bring the amino groups within 19 Å of one another in solution. These technical improvements allow this method to be used for investigating protein-protein interactions in complex biological samples.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA. funing.yan@einstein.yu.edu
authors.forename: Funing
authors.initials: F
authors.lastname: Yan
authors.forename: Fa-Yun
authors.initials: FY
authors.lastname: Che
authors.forename: Edward
authors.initials: E
authors.lastname: Nieves
authors.forename: Louis M
authors.initials: LM
authors.lastname: Weiss
authors.forename: Ruth H
authors.initials: RH
authors.lastname: Angeletti
authors.forename: Andras
authors.initials: A
authors.lastname: Fiser
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018360:Crystallography, X-Ray; D019281:Dimerization; D008954:Models, Biological; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D010455:Peptides