title: Isotope-coded and affinity-tagged cross-linking (ICATXL): an efficient strategy to probe protein interaction surfaces.
abstract: Chemical cross-linking followed by identification of the cross-linked residues by mass spectrometry provides structural information on protein interaction surfaces. Nevertheless, accurate analysis of the digested, cross-linked proteins is often challenging. Herein, we describe a novel strategy that relies on the use of affinity-tagged cross-linkers and isotope coding on the cross-linker-modified species. Incorporation of O16 or O18 during the hydrolysis of the cross-linkers results in a characteristic "doublet" for the undesired products of a half-cross-linking reaction. Therefore, genuine cross-linked peptides are readily distinguished for further structural analysis. This strategy permits a sensitive and facile analysis on a dimeric protease inhibitor, ecotin, showing general applicability to other protein assemblies.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
authors.forename: Feixia
authors.initials: F
authors.lastname: Chu
authors.forename: Sami
authors.initials: S
authors.lastname: Mahrus
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
mesh_terms: D000345:Affinity Labels; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D008958:Models, Molecular; D015394:Molecular Structure; D033903:Periplasmic Proteins / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D013499:Surface Properties
chemical_list: D000345:Affinity Labels; D003432:Cross-Linking Reagents; C039076:Eco protein, E coli; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D011506:Proteins