Chemical cross-linking and FTICR mass spectrometry for protein structure characterization

Abstract

Chemical cross-linking—a process forming covalent bonds between different molecules (intermolecular) or parts of a molecule (intramolecular)—has proven successful in combination with mass spectrometry [1, 2] as a tool for low-resolution structure determination [3, 4]. It is a fast procedure with low material consumption offering the opportunity to gain further insight into three-dimensional structures of proteins or protein complexes under native conditions. The goal of performing intramolecular chemical cross-linking of a protein is to get hints on its threedimensional structure [5], whereas the aim of intermolecular cross-linking between different proteins is to elucidate which components interact and how and where they physically contact each other [6]. Of the hundreds of cross-linking reagents described in the literature [7, 8] or offered commercially [9], most utilize common organic chemical principles that can be reduced to a few primary reactions. Homobifunctional cross-linking reagents contain identical functional groups at both reactive sites, which are connected with a carbon chain spacer bridging a defined distance. Therefore, identical functional groups in the proteins (e.g., amine or sulfhydryl groups) can be cross-linked. In contrast, heterobifunctional cross-linkers possess two different reactive sites. The number of cross-linking reagents has increased dramatically during the past 20 years, and today, a wide variety of reagents are commercially available possessing different spacer lengths and reactivities [9]. N-Hydroxysuccinimide (NHS) esters, targeting amine groups, are probably the most widely applied reagents for chemical cross-linking of proteins. As examples, Table 1 shows the two homobifunctional, water-soluble sulfoNHS esters sulfo-DST (sulfo-disuccinimidyl tartrate) and BS (bis(sulfosuccinimidyl)suberate). The smallest available reagent systems for chemical cross-linking are ‘zero-length’ cross-linkers, such as EDC (1-ethyl-3-(3dimethylaminopropyl)carbodiimide). These compounds mediate cross-linking between two proteins by creating a bond without an intervening linker and have proven beneficial especially for intermolecular cross-linking between proteins [6]. EDC is mostly applied in combination with N-hydroxysulfosuccinimide (sulfo-NHS) (Table 1). The advantage of adding sulfo-NHS to EDC consists in increasing the stability of the active intermediate, which ultimately reacts with the amine group. However, the cross-linking approach possesses some apparent limitations, since cross-linking conditions have to be specifically established for each protein or protein complex under investigation, and identification of crosslinking products is often a laborious procedure due to the complexity of the created cross-linking reaction mixtures. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry [10] represents an attractive tool for analyzing cross-linking reaction mixtures. Its high mass accuracy serves as an additional constraint in the identification of cross-linking reaction products. Additionally advantageous is that FTICRMS allows a ‘gas-phase’ purification and subsequent fragmentation of cross-linking products in the ICR cell. In general, chemical cross-linking in combination with FTICR mass spectrometry for protein structure characterization can be divided into two groups: