title: Reliable identification of cross-linked products in protein interaction studies by 13C-labeled p-benzoylphenylalanine.
abstract: We describe the use of the (13)C-labeled artificial amino acid p-benzoyl-L-phenylalanine (Bpa) to improve the reliability of cross-linked product identification. Our strategy is exemplified for two protein-peptide complexes. These studies indicate that in many cases the identification of a cross-link without additional stable isotope labeling would result in an ambiguous assignment of cross-linked products. The use of a (13)C-labeled photoreactive amino acid is considered to be preferred over the use of deuterated cross-linkers as retention time shifts in reversed phase chromatography can be ruled out. The observation of characteristic fragment ions additionally increases the reliability of cross-linked product assignment. Bpa possesses a broad reactivity towards different amino acids and the derived distance information allows mapping of spatially close amino acids and thus provides more solid structural information of proteins and protein complexes compared to the longer deuterated amine-reactive cross-linkers, which are commonly used for protein 3D-structure analysis and protein-protein interaction studies.
authors.affiliation: Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, 06120, Halle (Saale), Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Pettelkau
authors.forename: Christian H
authors.initials: CH
authors.lastname: Ihling
authors.forename: Petra
authors.initials: P
authors.lastname: Frohberg
authors.forename: Lars
authors.initials: L
authors.lastname: van Werven
authors.forename: Olaf
authors.initials: O
authors.lastname: Jahn
authors.forename: Andrea
authors.initials: A
authors.lastname: Sinz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001577:Benzophenones / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes / Q000032:analysis; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D025941:Protein Interaction Mapping / Q000379:methods; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: C488060:4-benzoylphenylalanine; D001577:Benzophenones; D002147:Calmodulin; D002247:Carbon Isotopes; D003432:Cross-Linking Reagents; D051604:Guanylate Cyclase-Activating Proteins; D010455:Peptides; D011506:Proteins; D010649:Phenylalanine