title: Enrichment of Cross-Linked Peptides Using Charge-Based Fractional Diagonal Chromatography (ChaFRADIC).
abstract: Chemical cross-linking of proteins is an emerging field with huge potential for the structural investigation of proteins and protein complexes. Owing to the often relatively low yield of cross-linking products, their identification in complex samples benefits from enrichment procedures prior to mass spectrometry analysis. So far, this is mainly accomplished by using biotin moieties in specific cross-linkers or by applying strong cation exchange chromatography (SCX) for a relatively crude enrichment. We present a novel workflow to enrich cross-linked peptides by utilizing charge-based fractional diagonal chromatography (ChaFRADIC). On the basis of two-dimensional diagonal SCX separation, we could increase the number of identified cross-linked peptides for samples of different complexity: pure cross-linked BSA, cross-linked BSA spiked into a simple protein mixture, and cross-linked BSA spiked into a HeLa lysate. We also compared XL-ChaFRADIC with size exclusion chromatography-based enrichment of cross-linked peptides. The XL-ChaFRADIC approach is straightforward, reproducible, and independent of the cross-linking chemistry and cross-linker properties.
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund 44227, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Tinnefeld
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund 44227, Germany.
authors.forename: A Saskia
authors.initials: AS
authors.lastname: Venne
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund 44227, Germany.|Department of Chemistry, College of Physical Sciences, University of Aberdeen , Aberdeen AB24 3UE, United Kingdom.|Medizinisches Proteom Center, Ruhr Universität Bochum , Bochum 44801, Germany.
authors.forename: Albert
authors.initials: A
authors.lastname: Sickmann
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund 44227, Germany.
authors.forename: René P
authors.initials: RP
authors.lastname: Zahedi
mesh_terms: D000818:Animals; D002417:Cattle; D002845:Chromatography / Q000379:methods*; D002852:Chromatography, Ion Exchange; D045424:Complex Mixtures / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D059748:Proteolysis; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification*; D055672:Static Electricity; D013388:Succinimides / Q000737:chemistry*
chemical_list: D045424:Complex Mixtures; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D013388:Succinimides; D012710:Serum Albumin, Bovine; C035760:bis(sulfosuccinimidyl)suberate; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase
keywords: ChaFRADIC; enrichment; mass spectrometry; protein cross-linking; strong cation exchange chromatography