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

App C, Knop J, Huff T, Seebahn A, Becker CM, Iavarone F, Castagnola M, Hannappel E (2014)

Publication Type: Journal article

Publication year: 2014

Journal

Publisher: Elsevier Masson

Book Volume: 456

Pages Range: 14-21

DOI: 10.1016/j.ab.2014.04.003

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.

Authors with CRIS profile

Additional Organisation(s)

Involved external institutions

Catholic University of the Sacred Heart / Università Cattolica del Sacro Cuore Italy (IT)

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://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.

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