Integrated Analysis of Cross-Links and Dead-End Peptides for Enhanced Interpretation of Quantitative XL-MS

Chemical cross-linking with mass spectrometry provideslow-resolutionstructural information on proteins in cells and tissues. Combinedwith quantitation, it can identify changes in the interactome betweensamples, for example, control and drug-treated cells or young andold mice. A difference can originate from protein conformational changesthat alter the solvent-accessible distance separating the cross-linkedresidues. Alternatively, a difference can result from conformationalchanges localized to the cross-linked residues, for example, alteringthe solvent exposure or reactivity of those residues or post-translationalmodifications of the cross-linked peptides. In this manner, cross-linkingis sensitive to a variety of protein conformational features. Dead-endpeptides are cross-links attached only at one end to a protein withthe other terminus being hydrolyzed. As a result, changes in theirabundance reflect only conformational changes localized to the attachedresidue. For this reason, analyzing both quantified cross-links andtheir corresponding dead-end peptides can help elucidate the likelyconformational changes giving rise to observed differences in cross-linkabundance. We describe analysis of dead-end peptides in the XLinkDBpublic cross-link database and, with quantified mitochondrial dataisolated from failing heart versus healthy mice, show how a comparisonof abundance ratios between cross-links and their corresponding dead-endpeptides can be leveraged to reveal possible conformational explanations.