title: Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer.
abstract: A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β4 can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI-MS (electrospray ionization-mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β4 did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β4 as well as of other proteins and peptides.
authors.affiliation: Institut für Biochemie, Emil Fischer Zentrum, Friedrich Alexander Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
authors.forename: Christine
authors.initials: C
authors.lastname: App
authors.affiliation: Institut für Biochemie, Emil Fischer Zentrum, Friedrich Alexander Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Knop
authors.affiliation: Institut für Biochemie, Emil Fischer Zentrum, Friedrich Alexander Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Huff
authors.affiliation: Institut für Biochemie, Emil Fischer Zentrum, Friedrich Alexander Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
authors.forename: Angela
authors.initials: A
authors.lastname: Seebahn
authors.affiliation: Institut für Biochemie, Emil Fischer Zentrum, Friedrich Alexander Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.
authors.forename: Cord-Michael
authors.initials: CM
authors.lastname: Becker
authors.affiliation: Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina, Università Cattolica, 00168 Roma, Italy; Istituto per la Chimica del Riconoscimento Molecolare-CNR, 20131 Milano, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Iavarone
authors.affiliation: Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina, Università Cattolica, 00168 Roma, Italy; Istituto per la Chimica del Riconoscimento Molecolare-CNR, 20131 Milano, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Castagnola
authors.affiliation: Institut für Biochemie, Emil Fischer Zentrum, Friedrich Alexander Universität Erlangen-Nürnberg, 91054 Erlangen, Germany. Electronic address: eh@biochem.uni-erlangen.de.
authors.forename: Ewald
authors.initials: E
authors.lastname: Hannappel
mesh_terms: D000199:Actins / Q000378:metabolism; D000588:Amines / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000737:chemistry*; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000737:chemistry*; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D008969:Molecular Sequence Data; D055668:Photochemical Processes; D011485:Protein Binding; D051381:Rats; D013194:Staining and Labeling; D013947:Thymosin / Q000737:chemistry* / Q000378:metabolism*; D011503:Transglutaminases / Q000378:metabolism; D014466:Ultraviolet Rays; D000099201:Thymosin beta(4)
chemical_list: D000199:Actins; D000588:Amines; D001386:Azides; D001710:Biotin; D002103:Cadaverine; D003432:Cross-Linking Reagents; D013947:Thymosin; D011503:Transglutaminases; D000099201:Thymosin beta(4); C000594174:cadaverine-2-(6-(biotinamido)-2-(p-azidobenzamido)hexanoamido)ethyl-1,3'-dithiopropionate
keywords: Actin; Cadaverine; Label transfer; Sulfo-SBED; Thymosin β(4); Transglutaminase