Česká společnost pro hmotnostní spektrometrii

(Česká konference hmotnostní spektrometrie 2017 - PL-1)
The power of cross-linking/mass spectrometry for protein structure analysis

Andrea Sinz ^1 *

1. Martin Luther University Halle-Wittenberg

Abstrakt

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. 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-linking data, three-dimensional structural models of proteins and protein complexes can be constructed.
One of our goals is to extend the arsenal of existing cross-linkers to obtain complementary structural information of proteins. To facilitate the identification of cross-linked products, we have designed MS cleavable cross-linkers creating characteristic marker ions in the fragment ion mass spectra [1]. We have evaluated different fragmentation methods available on an Orbitrap Fusion in combination with a dedicated software tool, MeroX, for conducting fully automated analyses of cross-linked products. An alternative, more exploratory strategy relies on a novel cross-linking reagent, containing a TEMPO and a benzyl group. The aim for designing this cross-linker was to facilitate the assignment of cross-linked products by FRIPS [2].
A direct way to probe protein-protein interactions is by site-specific incorporation of genetically encoded photo-reactive amino acids or by non-directed incorporation of photo-reactive amino acids. These photo-reactive amino acids contain benzophenone or diazirine groups and are activated upon UV-A irradiation [3]. As such, they possess the potential to study in vivo protein interactions.

* Korespondující autor: andrea.sinz@pharmazie.uni-halle.de

Literatura

1. Müller MQ. et al: Anal. Chem. 82, 6958-6968 (2010).
2. Hage C. et al: J. Am. Soc. Mass Spectrom. 28, 56-68 (2017).
3. Piotrowski C. et al: Methods 89, 121-127 (2015).