Abstract
Cross-linking mass spectrometry (XL-MS) has matured into a potent tool to identify protein–protein interactions or to uncover protein structures in living cells, tissues, or organelles. The unique ability to investigate the interplay of proteins within their native environment delivers valuable complementary information to other advanced structural biology techniques. This Review gives a comprehensive overview of the current possible applications as well as the remaining limitations of the technique, focusing on cross-linking in highly complex biological systems like cells, organelles, or tissues. Thanks to the commercial availability of most reagents and advances in user-friendly data analysis, validation, and visualization tools, studies using XL-MS can, in theory, now also be utilized by nonexpert laboratories.
Highlights
In the last few decades, cross-linking mass spectrometry (XLMS) has evolved into a widely accepted tool in structural biology
Most proteins are organized in multiprotein complexes with a tightly regulated structure that has a significant impact on their functions
For investigations of protein interaction topologies, approaches such as yeast two-hybrid (Y2H) systems,[2] proximityenhanced biotin labeling strategies (e.g., BioID3 or APEX4), immune precipitation, and affinity purification coupled to mass spectrometry are already commonly used and are reviewed elsewhere.[5,6]
Summary
In the last few decades, cross-linking mass spectrometry (XLMS) has evolved into a widely accepted tool in structural biology. By applying PhoX to a human cell lysate, more than 1100 cross-linked sites were successfully identified in a single measurement after IMAC enrichment This shows that the phosphonate group is highly applicable for an effective enrichment from complex samples, the data obtained with PhoX was searched against a reduced fasta file containing only the most abundant proteins to tackle the n2 problem.[44]. Depending on the search engine used, lists of cross-linked sites, cross-linked peptides, and monolinked peptides will be generated in a specific format Whereas some tools, such as MeroX, directly provide limited visualization options, like showing distance constraints compared with a Protein Data Bank (PDB) structure or showing interprotein cross-links within a network graph, the data is usually exported and processed by a different software for rearrangement, validation, or graphic visualizations. Confident links can be used to model protein structures or interaction sites or to generate PPI networks
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