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Development of comprehensive and user-friendly bioinformatics tools to study protein structures and interactions in mass spectrometry-based chemical cross-linking

Periodic Reporting for period 1 - BiT-XLMS (Development of comprehensive and user-friendly bioinformatics tools to study protein structures and interactions in mass spectrometry-based chemical cross-linking)

Reporting period: 2019-09-01 to 2021-08-31

Crosslinking combined with mass spectrometry (XLMS) provides information about protein structures, conformations as well as protein interactions. In recent years, the field has undergone remarkable developments both experimentally with a variety of novel crosslinkers, a chemical agent to capture spatially close residues on proteins, as well as computationally, to extend searching capabilities from a couple of proteins to a proteome-wide. Despite this, comprehensive and user-friendly tools which also quickly and efficiently address new MS technologies were limited thus preventing widespread application of XLMS.
This project aimed to develop comprehensive and user-friendly bioinformatics tools in XLMS and resulted in introducing MaxLynx (Yılmaz, et al., 2022), a novel computational proteomics workflow for XLMS integrated into the MaxQuant environment (Cox, et al., 2008). It can identify MS/MS spectra generated by two different crosslinker types: either non-cleavable or MS-cleavable crosslinkers. MaxLynx can be easily configured to search datasets generated by any novel chemical crosslinkers. It is user-friendly, fully automated and freely available. MaxLynx can run on Windows and Linux. It provides an interactive viewer to display annotated cross-linked spectra. Scientists from mass spectrometry or structural biology laboratories can analyze their XLMS data efficiently by using MaxLynx, without any assistance of bioinformatics experts. MaxLynx now will play an important role to make cross-linking one of the routine tools in structural biology.
The main outcome of this project is MaxLynx, a new crosslinking workflow integrated into MaxQuant (Cox, et al., 2008). Now any MaxQuant user can identify their XLMS spectra. To address this, Andromeda peptide database search engine (Cox, et al., 2011) was generalized to efficiently identify cross-linked peptides. In a typical MaxQuant workflow, after Andromeda score calculation, posterior error probabilities (PEP) are computed to control false-discovery rated (FDR). Here, PEPs are adapted for crosslinked peptides with introducing new parameters such as using partial scores summarize the evidence for the two constituents of the di-peptide individually. The three-dimensional peak detection of MaxQuant was improved to obtain more accurate determination of the monoisotopic peak of isotope patterns for heavy molecules, such as typical cross-linked peptides. A wide selection of filtering parameters is available which can replace manual filtering of identifications. On benchmark datasets of synthetic peptides, MaxLynx outperformed all other tested software on data for both types of crosslinkers as well as on a proteome-wide dataset of cross-linked D. melanogaster cell lysate (Yılmaz, et al., 2022). The MaxLynx workflow also supports a new MS technology, trapped-ion-mobility mass spectrometry. I presented the details of MaxLynx at the annual MaxQuant Summer School and MaxLynx has been recently published (Yılmaz, et al., 2022).
The output of this project is MaxLynx, a new software module to analyze spectra at XLMS. It is already integrated into MaxQuant, one of the most-used proteomics software, which is available for free at https://www.maxquant.org/
As being fully automated and user-friendly, now many researchers can analyze their data sets easily and without any immediate assistance. The outcome of MaxLynx can be used as complementary to other structural approaches to elucidate new protein structures and conformations, and to understand how proteins interact to each other. This can be useful to understand diseases and biological pathways.
Cross-linked peptide search modes - Peak refinement - Interactive inspection