Isolation of cross-linked peptides by diagonal strong cation exchange chromatography for protein complex topology studies by peptide fragment fingerprinting from large sequence databases

• Published in: Journal of Chromatography A Vol. 1348; pp. 34 - 46
• Main Authors: Buncherd, Hansuk, Roseboom, Winfried, Ghavim, Behrad, Du, Weina, de Koning, Leo J., de Koster, Chris G., de Jong, Luitzen
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 27.06.2014; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Liquid; Cross-linking; Cross-Linking Reagents; Databases, Protein; Diagonal chromatography; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; HeLa Cells; Humans; Mass spectrometry; Nuclear extract; Peptide Mapping; Peptides - chemistry; Peptides - isolation & purification; Protein Structure, Tertiary; Proteins; Salts - chemistry; Structural proteomics; Diagonal chromatography; Nuclear extract; Structural proteomics; Cross-linking; Mass spectrometry; Tandem mass spectrometry; Chemical analysis; Two dimensional chromatography; Peptides; Uterine cervix; Ion exchange chromatography; Separation method; Qualitative analysis; Tumor cell; Human; Coupled method; Crosslinking; Hela cell line; Protein; Cell line; Analysis method; Peptide fragment; Proteomics; Isolation; Cation exchanger; Structural analysis
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2014.04.083

•First report of isolation of cross-linked peptides by diagonal strong cation exchange chromatography.•First report of cross-link identification by peptide fragment fingerprinting from the entire human sequence database.•Discovery of a novel gas phase cleavage reaction of cross-linked peptides. 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.