Protein interactions enable much more complex behavior than the sum of the individual protein parts would suggest and represents a level of biological complexity requiring full understanding when unravelling cellular processes. Cross-linking mass spectrometry has emerged as an attractive approach to study these interactions, and recent advances in mass spectrometry and data analysis software have enabled the identification of thousands of cross-links from a single experiment. The resulting data complexity is, however, difficult to understand and requires interactive software tools. Even though solutions are available, these represent an agglomerate of possibilities, and each features its own input format, often forcing manual conversion. Here we present Cross-ID, a visualization platform that links directly into the output of XlinkX for Proteome Discoverer but also plays well with other platforms by supporting a user-controllable text-file importer. The platform includes features like grouping, spectral viewer, gene ontology (GO) enrichment, post-translational modification (PTM) visualization, domains and secondary structure mapping, data set comparison, previsualization overlap check, and more. Validation of detected cross-links is available for proteins and complexes with known structure or for protein complexes through the DisVis online platform ( ). Graphs are exportable in PDF format, and data sets can be exported in tab-separated text files for evaluation through other software.
A key step in therapeutic and endogenous humoral antibody characterization is identifying the amino acid sequence. So far, this task has been mainly tackled through sequencing of B-cell receptor (BCR) repertoires at the nucleotide level. Mass spectrometry (MS) has emerged as an alternative tool for obtaining sequence information directly at the – most relevant – protein level. Although several MS methods are now well established, analysis of recombinant and endogenous antibodies comes with a specific set of challenges, requiring approaches beyond the conventional proteomics workflows. Here, we review the challenges in MS-based sequencing of both recombinant as well as endogenous humoral antibodies and outline state-of-the-art methods attempting to overcome these obstacles. We highlight recent examples and discuss remaining challenges. We foresee a great future for these approaches making de novo antibody sequencing and discovery by MS-based techniques feasible, even for complex clinical samples from endogenous sources such as serum and other liquid biopsies.
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