MS Annika 2.0 Identifies Cross-Linked Peptides in MS2–MS3-Based Workflows at High Sensitivity and Specificity

Cross-linking mass spectrometry has become a powerful tool for the identification of protein–protein interactions and for gaining insight into the structures of proteins. We previously published MS Annika, a cross-linking search engine which can accurately identify cross-linked peptides in MS2 spectra from a variety of different MS-cleavable cross-linkers. In this publication, we present MS Annika 2.0, an updated version implementing a new search algorithm that, in addition to MS2 level, only supports the processing of data from MS2–MS3-based approaches for the identification of peptides from MS3 spectra, and introduces a novel scoring function for peptides identified across multiple MS stages. Detected cross-links are validated by estimating the false discovery rate (FDR) using a target-decoy approach. We evaluated the MS3-search-capabilities of MS Annika 2.0 on five different datasets covering a variety of experimental approaches and compared it to XlinkX and MaXLinker, two other cross-linking search engines. We show that MS Annika detects up to 4 times more true unique cross-links while simultaneously yielding less false positive hits and therefore a more accurate FDR estimation than the other two search engines. All mass spectrometry proteomics data along with result files have been deposited to the ProteomeXchange consortium via the PRIDE partner repository with the dataset identifier PXD041955.


Crosslink search workflow design and search parameters
Global parameters used across all search engines and all datasets: • Max.A more detailed description of the specific parameters in MS Annika/MS Annika 2.0 is given in the MS Annika manual that can be found here: https://github.com/hgb-bin-proteomics/MSAnnikaFor a more detailed overview of how the doublet detection and MS2 search works in MS Annika/MS Annika 2.0, please refer to the MS Annika 1.0 publication: https://doi.org/10.1021/acs.jproteome.0c01000S1 and Supplementary Figure S1 show the exact parameters and workflow used for crosslink identification.

Supplementary Table S1
• Dataset A Tolerances denoted with a slash are orbitrap tolerance/ion trap tolerance

Fig. S2:
A typical MS2-MS3 cross-linking workflow using a cleavable crosslinker.A crosslinker is added to the sample to covalently link residues in close spatial proximity forming crosslinks either within the same protein (intralink) or within different proteins (interlink).After digestion the cross-linked proteins are broken up into several cross-linked peptides that are analysed with LC-MS.In the MS1 scan the two peptides are still cross-linked together, yielding a peak of the whole cross-linked entity.For the MS2 scan, the cross-linked peptides are fragmented, the crosslinker is cleaved and the two peptides are broken up.Because the crosslinker incorporates an off-centre cleavage site each peptide is modified with either the shorter fragment or the longer fragment of the crosslinker, resulting in doublet peaks (coloured in blue and red in the MS2 scan) that show a crosslinker specific mass difference (e.g., 32 Da for DSSO).The doublet peaks are usually referred to by their crosslinker modification (mass) and the corresponding peptide, for example alpha light for the leftmost blue doublet peak: doublet peak originates from the alpha peptide and the respective crosslinker modification is the short fragment which yields a smaller mass, therefore it is the light peak (in comparison to the longer fragment that yields the heavy peak).For doublet peaks an MS3 scan is recorded after the MS2 scan where each MS3 scan should only contain one of the two cross-linked peptides in linear form.

Fig. S3 :
Fig.S3: Fragmentation behaviour of DSSO1 (exemplary also for other sulfoxy-based crosslinkers like DSBSO).During fragmentation the two peptides cross-linked with DSSO (top left) are cleaved which results in the beta peptide carrying the alkene fragment (+54 Da) and the alpha peptide carrying the sulfenic acid fragment (+104 Da) (top right) and vice versa (not depicted).This pairing of a peptide modified with alkene and a peptide modified with sulfenic acid results in a doublet with a mass difference of 50 Da, as depicted in the MS2 scan in the bottom right.However, during fragmentation the sulfenic acid fragment might lose a water molecule, transforming it into the thiol fragment instead.This creates a pairing of peptide modified with the alkene fragment (+54 Da) and peptide modified with the thiol fragment (+86 Da), resulting in a different doublet with a mass difference of 32 Da, as depicted in the bottom left.Detection of both cases by the search engine is essential for the correct calculation of the unmodified peptide's mass which subsequently is important for peptide identification.Graphics of DSSO are taken from the publication by Kao et al 1 .
All of the scripts use Microsoft Excel files as input, for that MS Annika results (any MS Annika version works) need to be exported from Proteome Discoverer by opening the "crosslink" result table and then select "File > Export > To Microsoft Excel… > Level 1: Crosslinks > Export" • result.xlsx-MS Annika result file(s) exported to .xlsx• seq.fasta -FASTA file containing sequences of the crosslinked proteins "python xiNetExporter_msannika.py result.xlsx-fasta seq.fasta"Exporting to xiVIEW: Files needed: • result.xlsx-MS Annika result file(s) exported to .xlsx• seq.fasta -FASTA file containing sequences of the crosslinked proteins "python xiViewExporter_msannika.py result.xlsx-fasta seq.fasta"Exporting to pyXlinkViewer (pyMOL):