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.

Unexpected Complexity of the Ammonia Monooxygenase in Archaea

“…To identify cross-linked peptides, the raw data were searched with either MS Annika 96 in Proteome Discoverer 2.3 or with MeroX 2.0 97 against the sequences of the top abundant protein hits (with at least 10 MS/MS counts) from the MaxQuant search. Although it had less than 10 MS/MS counts, the protein encoded by NVIE_004550 was also added based on other proteomic and syntenic analysis.…”

Mimicked synthetic ribosomal protein complex for benchmarking crosslinking mass spectrometry workflows

“…As representatively shown on the data generated with DSSO the benchmarked search engines all output higher real FDRs than the estimated 1% on XL level (Figure 1A). For this dataset MS Annika 23 and MaxLynx 24 perform best, both by means of correct FDR estimation as well as by means of unique ID numbers. We additionally applied post-scorecutoffs to correct the real FDR to ≤ 1%.…”

“…Figure 1: Benchmarking of data analysis tools on the example of DSSO. (A) Average XL ID numbers using DSSO applied to the main library and the algorithms MeroX 25 , Annika 23 , XlinkX 26 , pLink2 27 or MaxLynx 24 for analysis. All results were obtained at 1% estimated FDR and corrected by applying a post-score cutoff to reach a real FDR<=1.…”

Exhaustive Cross-linking Search with Protein Feedback

“…All the peptide masses in the various situations are calculated by the weighted-average mass of the detected peaks. Table S1 summarizes the strategies that MS Annika [6], XlinkX, Maxlynx [7], MeroX RISE mode, and ECL-PF adopt based on Figure S2. ECL-PF thus adopts the most comprehensive scheme to find the potential α/β peptide mass region.…”

“…These approaches try to take advantage of the hardware. From the perspective of software development, however, only a handful of work has been done so far [16,[20][21][22][23][24], and the sensitivity is still in the range of 20% ∼ 50% (observed from simulated datasets). We have discovered that using proteinpeptide association during the identification process improves the sensitivity considerably.…”