Cross-linking/Mass Spectrometry: A Community-Wide, Comparative Study Towards Establishing Best Practice Guidelines

Abstract

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has largely increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats that are generally accepted in the field and that have shown to lead to high-quality results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. From the results obtained, common protocols will be established. Our study serves as basis for establishing best practice guidelines in the field for conducting cross-linking experiments, performing data analysis, and reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

Optimized Fragmentation Improves the Identification of Peptides Cross-Linked by MS-Cleavable Reagents

“…In the case of BSA, the newly developed stepped-HCD method identified an average of 75 unique cross-linked sites, statistically not significantly more than MS 2 –MS 3 , which identified an average of 71. These results are in the same magnitude as suggested by a community-wide XLMS study . However, both outperformed the approach employing two complementary fragmentation types CID-ETD by 56 and 48%, respectively (Figure A).…”

“…These results are in the same magnitude as suggested by a community-wide XLMS study. 26 However, both outperformed the approach employing two complementary fragmentation types CID-ETD by 56 and 48%, respectively (Figure 6A). Therefore, this approach was not included for the comparison of the more complex sample.…”

xiView: A common platform for the downstream analysis of Crosslinking Mass Spectrometry data

“…Across the crosslinking community, it is accepted that "the term 'cross-link' refers to the specific amino acid residues that are connected, irrespective of different peptide sequences due to missed cleavage sites or modifications." 12 A PSM provides a single distance restraint by identifying a pair of crosslinked peptides. Typically, this has been in one spectrum, though isotope labelling and multiple fragmentation techniques can mean a PSM is associated with more than one spectrum (see Section 5.2.9 of the MzIdentML 1.2 specification).…”