Heterobifunctional isotope-labeled amine-reactive photo-cross-linker for structural investigation of proteins by matrix-assisted laser desorption/ionization tandem time-of-flight and electrospray ionization LTQ-Orbitrap mass spectrometry.
Chemical cross-linking combined with a subsequent enzymatic digestion and mass spectrometric analysis of the created cross-linked products presents an alternative approach to assess low-resolution protein structures. By covalently connecting pairs of functional groups within a protein or a protein complex a set of structurally defined interactions is built up. We synthesized the heterobifunctional amine-reactive photo-cross-linker N-succinimidyl p-benzoyldihydrocinnamate as a non-deuterated (SBC) and doubly deuterated derivative (SBDC). Applying a 1:1 mixture of SBC and SBDC for cross-linking experiments aided the identification of cross-linked amino acids in the mass spectra based on the characteristic isotope patterns of fragment ions. The cross-linker was applied to the calcium-binding protein calmodulin with a subsequent analysis of cross-linked products by nano-high-performance liquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (nano-HPLC/MALDI-TOF/TOF-MS) and nano-HPLC/nano-electrospray ionization (ESI)-LTQ-Orbitrap-MS.
Krauth
Fabian
F
Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle (Saale), Germany.
Ihling
Christian H
CH
Rüttinger
Hans Hermann
HH
Sinz
Andrea
A
D000818:Animals; D001577:Benzophenones / Q000138:chemical synthesis / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D007553:Isotope Labeling; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013388:Succinimides / Q000138:chemical synthesis / Q000737:chemistry*
D001577:Benzophenones; D002147:Calmodulin; D003432:Cross-Linking Reagents; C096731:N-succinimidyl 4-benzoyldihydrocinnamate; D013388:Succinimides