Improving cross-linking mass spectrometry methodologies

Research regarding protein interactions has been a productive area of study over many decades, contributing to our understanding of biology and health and hence to the development of therapies. Since most traditional methods are biased towards purified proteins with a fixed structure or a relatively low molecular mass, novel methods are required to monitor protein interactions in a wider range of situations. Cross-linking mass spectrometry (XL-MS) is emerging as a widely applicable technique that captures dynamic situations in protein complexes, and can readily be applied to endogenous levels of protein due to its high sensitivity. Since digested peptides are measured rather than the intact protein complex, XL-MS can theoretically be applied to proteins of any size. While an XL-MS experiment is relatively straight-forward in terms of experimental design, identification of cross-linked peptides via mass spectrometry remains challenging. Difficulties in optimising the ideal settings during such measurements arise from the variability of biological samples containing cross-linked peptides. We therefore aim to build a library of synthetic cross-linked peptides that will be used for systematic optimisation of MS parameters used in XL-MS studies. A further complication lies in the bioinformatics approaches that are used to identify cross- linked peptides from the MS data because two peptides need to be correctly identified rather than just one, as in other proteomics experiments. Additionally, it is never known exactly which cross-linked peptides are present in the digest of a cross-linked protein, since non- specific cross-links cannot be fully eliminated. We will therefore use the data collected in this study to create a dataset suitable for testing and optimising search engines that have been developed for the identification of cross-links from MS data. The use of synthetic peptides is particularly applicable in this case since the library can be controlled for true positives and true negatives, which is not possible upon analysis of a complex tryptic digestion mixture. We will also develop a standardised cross-linking experiment to track improvements in the full XL-MS workflow upon optimisation of MS parameters. The standardised experiment will be based on cross-linking of the Cas9 protein. Such a standardised is imperative for the cross-linking community to compare the quality of data obtained from different methodologies. Improvements that will be made to the XL-MS technique as a result of this research will enable more protein interaction sites to be discovered and characterised, adding to the biological impact of such experiments and ensuring that cross-linked peptides are correctly identified.

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