MS Annika: A New Cross-Linking Search Engine

Abstract

Cross-linking mass spectrometry (XL-MS) has become a powerful technique that enables insights into protein structures and protein interactions. The development of cleavable cross-linkers has further promoted XL-MS through search space reduction, thereby allowing for proteome-wide studies. These new analysis possibilities foster the development of new cross-linkers, which not every search engine can deal with out of the box. In addition, some search engines for XL-MS data also struggle with the validation of identified cross-linked peptides, that is, false discovery rate (FDR) estimation, as FDR calculation is hampered by the fact that not only one but two peptides in a single spectrum have to be correct. We here present our new search engine, MS Annika, which can identify cross-linked peptides in MS2 spectra from a wide variety of cleavable cross-linkers. We show that MS Annika provides realistic estimates of FDRs without the need of arbitrary score cutoffs, being able to provide on average 44% more identifications at a similar or better true FDR than comparable tools. In addition, MS Annika can be used on proteome-wide studies due to fast, parallelized processing and provides a way to visualize the identified cross-links in protein 3D structures.

Keywords: MS/MS; PPI; XL-MS; bioinformatics; cross-linking; protein-protein-interaction; search engine; tandem mass spectrometry.

Figure 1

MS Annika search engine. The MS Annika Detector identifies potential ion doublets and passes the spectra to the search node if doublets are found. Spectra with identified ion doublets are searched using the MS Annika Search node. The final combination of CSM matches to cross-links as well as multilevel FDR control fall into the scope of the MS Annika Validator node. The optional xiView Exporter node can be used to export cross-links at different confidence levels, which can then be uploaded to xiView for evaluation.

Figure 2

Cross-links at one and five percent estimated FDRs and calculated FDRs for four different tools, namely, MeroX, XlinkX, pLink, and MS Annika. Searches have been performed on the data set provided by Beveridge and co-workers, consisting of synthetic peptides in multiple groups, where peptides within a group are cross-linked. False-positive XLs (dark orange) are cross-linked peptides in different groups, enabling the comparison between the estimated FDR (based on a target-decoy search) and the calculated FDR based on the false-positive XLs. Calculated FDRs are generally higher than the estimated FDRs. Results shown were obtained using no score cutoff for any of the tools. For XlinkX, a minimum score difference (delta score) of 4 was set. Score cutoffs can often remove many true positives and can obfuscate the selection of cross-links. In PD 2.5, a new FDR calculation strategy was introduced for XlinkX.

Figure 3

Unique cross-link counts for measurements from the CIDMS2-HCDMS2 acquisition mode of the data set created by Ser et al. All three tools were set to 1% estimated FDR without additional score cutoffs. While MeroX and XlinkX both provide more results, large fractions of identified cross-links are non-BSA cross-links (red). When applying a 1% postsearch score cutoff (removing the lowest-scoring item until 1% or less are incorrect items), only the blue items are retained. MS Annika properly separates true and false positives and provides only correct identifications.

Figure 4

Visualization in xiView of DSSO cross-links in the data set created by Stieger et al. and identified by MS Annika mapped to the 3D structure of E. coli ribosomes (PDB identifier 5IT8), displaying only cross-links shorter than 26 Å. Orange cross-links connect two residues of the same protein (intralinks), while blue cross-links span residues between different proteins (interlinks).