Chemical crosslinking in combination with mass spectrometry has matured into an alternative approach to derive low-resolution structural information of proteins and protein complexes. Yet, one of the major drawbacks of this strategy remains the lack of software that is able to handle the large MS datasets that are created after chemical crosslinking and enzymatic digestion of the crosslinking reaction mixtures. Here, we describe a software, termed StavroX, which has been specifically designed for analyzing highly complex crosslinking datasets. The StavroX software was evaluated for three diverse biological systems: (1) the complex between calmodulin and a peptide derived from Munc13, (2) an N-terminal ß-laminin fragment, and (3) the complex between guanylyl cyclase activating protein-2 and a peptide derived from retinal guanylyl cyclase. We show that the StavroX software is advantageous for analyzing crosslinked products due to its easy-to-use graphical user interface and the highly automated analysis of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data resulting in short times for analysis. StavroX is expected to give a further push to the chemical crosslinking approach as a routine technique for protein interaction studies.
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.
The electrochemical oxidation of various open-chain and cyclic amidrazones in acetonitrile was investigated by cyclic voltammetry. The oxidation was found reversible for both N 2 -disubstituted open-chain amidrazones and all cyclic compounds with the exception of triazole derivatives that could not be oxidized. Microcoulometry revealed an extrapolated charge consumption of one electron per molecule. The experimentally obtained oxidation potentials correlate well with the reaction energy of oxidation calculated from density function theory (DFT) that clearly supports the hypothesis of a persistent radical formation. The total atomic spin density of radical cations was calculated and permits to make a statement about the localization of radical formation. The synthesis of (E)-2-(methylphenyl)hydrazono-N-phenyl-2-piperidin-1-ylacetamide 7 representing open-chain α-carbonyl substituted N 2 -alkyl, aryl-amidrazones and the synthesis of 6-aminosubstituted 2,3,4,5-tetrahydro-1,2,4-triazin-5-ones, a new class of amidrazones, are described.
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