title: Isolation of cross-linked peptides by diagonal strong cation exchange chromatography for protein complex topology studies by peptide fragment fingerprinting from large sequence databases.
abstract: Knowledge of spatial proximity of amino acid residues obtained by chemical cross-linking and mass spectrometric analysis provides information about protein folding, protein-protein interactions and topology of macromolecular assemblies. We show that the use of bis(succinimidyl)-3-azidomethyl glutarate as a cross-linker provides a solution for two major analytical problems of cross-link mapping by peptide fragment fingerprinting (PFF) from complex sequence databases, i.e., low abundance of protease-generated target peptides and lack of knowledge of the masses of linked peptides. Tris(carboxyethyl)phosphine (TCEP) reduces the azido group in cross-linked peptides to an amine group in competition with cleavage of an amide bond formed in the cross-link reaction. TCEP-induced reaction products were separated by diagonal strong cation exchange (SCX) from unmodified peptides. The relation between the sum of the masses of the cleavage products and the mass of the parent cross-linked peptide enables determination of the masses of candidate linked peptides. By reversed phase LC-MS/MS analysis of secondary SCX fractions, we identified several intraprotein and interprotein cross-links in a HeLa cell nuclear extract, aided by software tools supporting PFF from the entire human sequence database. The data provide new information about interacting protein domains, among others from assemblies involved in splicing.
authors.affiliation: Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 HX Amsterdam, The Netherlands.
authors.forename: Hansuk
authors.initials: H
authors.lastname: Buncherd
authors.affiliation: Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 HX Amsterdam, The Netherlands.
authors.forename: Winfried
authors.initials: W
authors.lastname: Roseboom
authors.affiliation: Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 HX Amsterdam, The Netherlands.
authors.forename: Behrad
authors.initials: B
authors.lastname: Ghavim
authors.affiliation: Van't Hoff Institute of Molecular Sciences, University of Amsterdam, Science Park 904, 1098 HX Amsterdam, The Netherlands.
authors.forename: Weina
authors.initials: W
authors.lastname: Du
authors.affiliation: Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 HX Amsterdam, The Netherlands.
authors.forename: Leo J
authors.initials: LJ
authors.lastname: de Koning
authors.affiliation: Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 HX Amsterdam, The Netherlands.
authors.forename: Chris G
authors.initials: CG
authors.lastname: de Koster
authors.affiliation: Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 HX Amsterdam, The Netherlands. Electronic address: l.dejong@uva.nl.
authors.forename: Luitzen
authors.initials: L
authors.lastname: de Jong
mesh_terms: D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents; D030562:Databases, Protein; D006367:HeLa Cells; D006801:Humans; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D012492:Salts / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D010455:Peptides; D012492:Salts
keywords: Cross-linking; Diagonal chromatography; Mass spectrometry; Nuclear extract; Structural proteomics