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  • Title: Cleavable cross-linker for protein structure analysis: reliable identification of cross-linking products by tandem MS.
    Author: Müller MQ, Dreiocker F, Ihling CH, Schäfer M, Sinz A.
    Journal: Anal Chem; 2010 Aug 15; 82(16):6958-68. PubMed ID: 20704385.
    Abstract:
    Chemical cross-linking combined with a subsequent enzymatic cleavage of the created cross-linked complex and a mass spectrometric analysis of the resulting cross-linked peptide mixture presents an alternative approach to high-resolution analysis, such as NMR spectroscopy or X-ray crystallography, to obtain low-resolution protein structures and to gain insight into protein interfaces. Here, we describe a novel urea-based cross-linker, which allows distinguishing different cross-linking products by collision-induced dissociation (CID) tandem MS experiments based on characteristic product ions and constant neutral losses. The novel cross-linker is part of our ongoing efforts in developing collision-induced dissociative reagents that allow an efficient analysis of cross-linked proteins and protein complexes. Our innovative analytical concept is exemplified for the Munc13-1 peptide and the recombinantly expressed ligand binding domain of the peroxisome proliferator-activated receptor alpha, for which cross-linking reaction mixtures were analyzed both by offline nano-HPLC/MALDI-TOF/TOF mass spectrometry and by online nano-HPLC/nano-ESI-LTQ-orbitrap mass spectrometry. The characteristic fragment ion patterns of the novel cross-linker greatly simplify the identification of different cross-linked species, namely, modified peptides as well as intrapeptide and interpeptide cross-links, from complex mixtures and drastically reduce the potential of identifying false-positive cross-links. Our novel urea-based CID cleavable cross-linker is expected to be highly advantageous for analyzing protein 3D structures and protein-protein complexes in an automated manner.
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