During the last 15 years, chemical cross-linking combined with mass spectrometry (MS) and computational modeling has advanced from investigating 3D-structures of isolated proteins to deciphering protein interaction networks [1,2]. Chemical cross-linking relies on the introduction of a covalent bond between functional groups of amino acids within one protein, to gain insight into the conformation of a protein, or between interaction partners to elucidate interfaces in protein complexes. Based on the distance restraints derived from the chemical cross-links, three-dimensional structural models of proteins and protein complexes can be constructed. Most commonly, homobifunctional amine-reactive cross-linkers, such as N-hydroxysuccinimide esters, are used for studying protein-protein interactions. One of our goals is to extend the arsenal of existing cross-linkers to obtain complementary 3D-structural information of proteins and protein complexes. To facilitate the identification of cross-linked products, we have designed novel MS/MS cleavable cross-linkers creating characteristic marker ions upon fragmentation [3].

In my talk, I will describe an integrated workflow for the automated identification of cross-linked products based on an MS/MS cleavable cross-linker using the different fragmentation methods available on an Orbitrap Fusion mass spectrometer (CID, HCD, ETciD, and EThcD). For conducting fully automated analyses, we employ our in-house developed software tool MeroX [4]. A direct way to probe protein-protein interactions in vivo is by site-specific incorporation of genetically encoded photo-reactive amino acids or by non-directed incorporation of photo-reactive amino acids. I will illustrate the different cross-linking strategies based on two protein systems: The tetrameric tumor suppressor protein p53 [5] and the complex between the basement membrane proteins laminin and nidogen-1 [6].

Literature References:

[1] Sinz, A., Expert Rev. Proteomics 2014, 11, 733.[2] Herzog et al., Science 2012,337, 1348. [3] Müller, M.Q. et al., Anal. Chem. 2010, 82, 6958. [4] Götze, M. et al., J. Am. Soc. Mass Spectrom. 2015, 26, 83. [5] Arlt et al., Proteomics 2015,14, 3996. [6] Lössl et al., PLoSOne 2014, 9:e112886.