Merck
  • Home
  • Search Results
  • Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer.

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

Analytical biochemistry (2014-04-16)
Christine App, Jana Knop, Thomas Huff, Angela Seebahn, Cord-Michael Becker, Federica Iavarone, Massimo Castagnola, Ewald Hannappel
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cadaverine, 95%
Sigma-Aldrich
Acetonitrile solution, contains 0.05 % (v/v) trifluoroacetic acid
Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC
Sigma-Aldrich
Cadaverine, purum, ≥97.0% (GC)
Millipore
Acetonitrile solution, suitable for HPLC, acetonitrile:water 5:95% (v/v), 10 mM Ammoniumbicarbonate, pH 10,0
Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) formic acid, suitable for HPLC
Supelco
Cadaverine, analytical standard
Supelco
Residual Solvent - Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Fluorescamine, ≥98% (TLC), powder, used for detection of primary amines
Supelco
Acetonitrile, analytical standard
Sigma-Aldrich
Acetonitrile, for DNA synthesis
Sigma-Aldrich
Acetonitrile, Preparateur, ≥99.9% (GC), Customized plastic drum
Sigma-Aldrich
Acetonitrile, anhydrous, 99.8%
Supelco
Trifluoroacetic acid, analytical standard
Sigma-Aldrich
Acetonitrile, puriss. p.a., ACS reagent, ≥99.5% (GC)
Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Trifluoroacetic acid, suitable for HPLC, ≥99.0%
Sigma-Aldrich
Acetonitrile, HPLC Plus, ≥99.9%
Sigma-Aldrich
Trifluoroacetic acid, puriss. p.a., suitable for HPLC, ≥99.0% (GC)
USP
Residual Solvent Class 2 - Acetonitrile, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Ultrapure Acetonitrile
Sigma-Aldrich
Trifluoroacetic acid, ReagentPlus®, 99%
Sigma-Aldrich
Acetonitrile, ReagentPlus®, 99%
Supelco
Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Acetonitrile, suitable for HPLC-GC, ≥99.8% (GC)
Sigma-Aldrich
Acetonitrile, for HPLC, for UV, ≥99.9% (GC)
Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Acetonitrile, ACS reagent, ≥99.5%
Sigma-Aldrich
Acetonitrile, suitable for DNA synthesis, ≥99.9% (GC)
Sigma-Aldrich
Acetonitrile, ≥99.5% (GC)