Mascot: The trusted reference standard for protein identification by mass spectrometry for 25 years

Posted by Richard Jacob (December 5, 2023)

ABRF iPRG 2023 crosslinking study

One of the great things about the Association of Biomolecular Resource Facilities (ABRF) is their technical studies involving real samples and data. The three proteomics research groups, Proteomics (PRG), Proteome Informatics (iPRG) and Proteomics Standards (sPRG), have all held interesting studies over the years. This year the iPRG group has put together a crosslinking study. The goal is to teach people how to analyze crosslinked samples with existing software and provide them with data sets that they can practice on. It is then hoped that more core labs will offer crosslinking analysis as a service to customers and collaborators.

The study consists of recombinant proteins that were purified from an E. coli expression system. The expressed proteins form complexes under optimal conditions. The preparations were crosslinked with Disuccinimidyl sulfoxide (DSSO), ultracentrifuged and separated by SDS-PAGE.  Bands were excised before reduction and alkylation with iodoacetamide and digestion with trypsin. The digests were analyzed on a Thermo Scientific Orbitrap Fusion Lumos using both the low resolution ion trap and high resolution orbitrap acquisition methods.

Three data files have been used for tutorials on different software analysis tools as phase 1 of the study. Matrix Science has been fortunate to be invited to provide a tutorial (ABRF_iPRG_2022 study_XLink tutorial_Mascot.pdf) for the analysis using Mascot Server and Distiller.

In our tutorial, we provide a walk through of the steps required to analyze a crosslinked data set from configuring the FASTA database and crosslinking method to processing the mass spec data and reviewing the search results. The data is high resolution MS/MS data and we processed them with Mascot Distiller. Crosslinked peptides can have quite high masses which means they often have higher charge states too. We recommend either decharging the fragment ions to MH+ or exporting their charge state in the MGF file, both of which help a lot in the database search. We include alternative analysis pathways using Mascot Server directly, Mascot Distiller and Server and finally using an automated pipeline with Mascot Daemon, Distiller and Server.

As part of the ABRF iPRG study, we have added the iPRG protein sequences and crosslinking method to the public server. However, the public server has limits to the number of queries it can analyze in a single search. The data files for the iPRG study are too large for a public search. If you don’t have your own Mascot Server and would like to try searching this dataset on the public Mascot Server, please contact to request a temporary account with expanded limits.

The final part of the tutorial covers analysis of the results. We have previously published some guidelines for validating intact crosslinked peptide matches and you can use those guidelines to determine what a good match looks like and which ones don’t have sufficient information to validate the crosslink or are poor/random matches. With a large number of matches it can be difficult to see the relationship between the crosslinked matches due to multiple charge states, modifications and different peptide lengths. We find it easier to view the results sorted by crosslink site in specialized software designed for the task. Mascot Server results can be reviewed in xiVIEW.

We have made available some resources that allow you to jump in to the analysis at any step from raw data, to peak list, Mascot Server search results and xiVIEW.

If you have any questions about using Mascot Server for crosslinking analysis please let us know at


Tutorial using Mascot: ABRF_iPRG_2022 study_XLink tutorial_Mascot.pdf

Mascot Distiller raw data processing options: prof_prof.200.ThermoXcalibur.opt


MGF file

Mascot Server search result

xiVIEW project













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