A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry

Abstract

Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy[1]. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs. We report the adaptation of the widely used, open source software package Skyline, for the analysis of quantitative XL-MS data as a means for data analysis and sharing of methods. We demonstrate the utility and robustness of the method with a cross-laboratory study and present data that is supported by and validates previously published data on quantified cross-linked peptide pairs. This advance provides an easy to use resource so that any lab with access to a LC-MS system capable of performing targeted quantitative analysis can quickly and accurately measure dynamic changes in protein structure and protein interactions.

Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry

“…In the current study we identified crosslinked sites between murine POLRMT and the mitochondrial translation initiation factor IF-2 that are consistent with these proposed secondary functions (70). Last, each of the cross-linked peptide pairs identified here has the potential to serve as a molecular probe for the future study of dynamic in vivo protein conformations and interactions (35,71).…”

“…First, we generated two databases to enable (i) targeted quantitative analysis of protein-protein relationships by parallel reaction monitoring (PRM) (71) and (ii) large-scale identification of new datasets by spectral matching (Fig. S7) (27,72).…”

“…Importantly, neither PRM nor spectral library matching requires specialized instrumentation to identify and quantify XL-MS interactions, so these techniques can be transferred rapidly between laboratories and core facilities (71), allowing wide dissemination of XL-MSbased analysis of mitochondrial proteins. Second, to enable widespread use of these data by the community, we developed a flexible web tool (github.com/mammmals/crosslink-PRMtransitionForm) (71) to allow users to generate PRM transitions for any cross-linked peptide interaction identified in this study using any user-selected cross-linker or desired modification.…”

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

“…XL-MS technology is a powerful and effective structural tool because it offers several layers of information through the identification of cross-linked peptides 4,6,7,23,38,79,138,139 . Within a given protein complex, cross-linking analysis identifies proximal residues between subunits of protein complexes, providing clues on spatial orientation and protein connectivity.…”

“…Until now, only a limited number of in vivo XL-MS studies have been reported, mainly due to the challenges discussed above. While the Bruce lab has focused on in vivo cross-linking at the proteome level using their PIR cross-linkers 40,77,89,104–106,108,139,151 , our lab has mostly focused on in vivo XL-MS analysis of protein complexes 79,99,159,162,166,167 . Recently, we have developed a membrane permeable, sulfoxide-containing MS-cleavable and enrichable NHS ester cross-linker Azide-A-DSBSO (Table 2) 79 .…”