Cleavable Cross-Linkers and Mass Spectrometry for the Ultimate Task of Profiling Protein-Protein Interaction Networks in Vivo

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.

Keywords: acquisition techniques; cross-linking; fragmentation techniques; guideline; mass spectrometry.

Figure 1

Applications of XL-MS. A cross-linker consists of three main elements: First, the reactive group either targets specific amino acid residues or nonspecifically reacts with any amino acid. Second, the spacer arm might contain one or more labile sites for MS cleavability. Shorter spacers provide higher resolution structural data but will lead to fewer cross-links. Third, some reagents bear an enrichment handle for the selective capture of cross-linked peptides. The linker molecule can be applied either to single proteins/protein complexes (shown in green) or in vivo (shown in blue). After MS/MS acquisition and data analysis, the obtained cross-links can give valuable information on the protein structure, complex topologies, conformational changes, specific interaction sites, or (proteome-wide) protein–protein interaction (PPI) networks.

Figure 2

Fragmentation spectra of gas-phase cleavable cross-linkers. Upon collision-induced (CID/HCD) or electron-transfer-induced (ETD) dissociation, cleavable cross-linkers break apart, usually at two different positions. This leads to the formation of a characteristic doublet (MS2 level). Alternatively, in the case of protein interaction reporter (PIR) linkers, a reporter ion of a known mass is formed (shown as a red triangle). These ions can be used to unambiguously identify the presence of a cross-linked peptide, which is then selected for further fragmentation to obtain peptide fragments and to identify the amino acid sequence (MS3 level). In the case of stepped HCD or ETD fragmentation, this MS3 level is omitted; instead, the diagnostic cross-linker ions as well as the peptide fragments are detected in the same MS2 spectrum. Peptides are illustrated in green and blue, respectively, the cross-linker is shown in brown, and cleavage sites are shown as red dashed lines.

Figure 3

Schematic overview of the applied enrichment strategies in the field of cross-linking mass spectrometry. A combination of these techniques can improve the efficiency of cross-link isolation but also increases sample loss as a trade.

Figure 4

Comparison of MeroX in different modes and XlinkX upon the variation of database size. (A) DSBU cross-linked BSA was analyzed using MeroX (v 2.0.1.4, different modes) or XlinkX (within Thermo Proteome discoverer v 2.4) against a database containing BSA spiked with proteins of the human proteome to obtain total sizes of 1–10 000 proteins. Results were filtered at 5% FDR on the spectrum level. Bars indicate the number of unique cross-linked residue pairs: green, BSA (intra-) links; red, interlinks and non-BSA intralinks. (B) In addition, a score cutoff of 50 was applied to all MeroX results, and XlinkX data were filtered for a minimal score of 40 and a minimal delta score of 4. (C) Runtime needed for data analysis with the largest database containing 10 000 proteins.