Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer

Journal article


Publication Details

Author(s): App C, Knop J, Huff T, Seebahn A, Becker CM, Iavarone F, Castagnola M, Hannappel E
Journal: Analytical Biochemistry
Publisher: Elsevier Masson
Publication year: 2014
Volume: 456
Pages range: 14-21
ISSN: 0003-2697


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 ?4 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 ?4 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 ?4 as well as of other proteins and peptides.



FAU Authors / FAU Editors

App, Christine
Lehrstuhl für Biochemie und Pathobiochemie
Becker, Cord-Michael Prof. Dr.
Medizinische Fakultät
Hannappel, Ewald Prof. Dr.
Professur für Biochemie und Molekulare Neurowissenschaften
Huff, Thomas PD Dr.
Institut für Biochemie
Knop, Jana Dr.
Lehrstuhl für Biochemie und Pathobiochemie
Seebahn, Angela
Lehrstuhl für Biochemie und Molekulare Medizin


Additional Organisation
Emil-Fischer-Zentrum (Emil Fischer Center)


External institutions with authors

Catholic University of the Sacred Heart / Università Cattolica del Sacro Cuore


How to cite

APA:
App, C., Knop, J., Huff, T., Seebahn, A., Becker, C.-M., Iavarone, F.,... Hannappel, E. (2014). Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer. Analytical Biochemistry, 456, 14-21. https://dx.doi.org/10.1016/j.ab.2014.04.003

MLA:
App, Christine, et al. "Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer." Analytical Biochemistry 456 (2014): 14-21.

BibTeX: 

Last updated on 2018-08-10 at 02:49