Accurate and Automated High-Coverage Identification of Chemically Cross-Linked Peptides with MaxLynx.

Şule Yılmaz,Florian Busch,Jürgen Cox,Nagarjuna Nagaraj

doi:10.1021/acs.analchem.1c03688

Abstract

Cross-linking combined with mass spectrometry (XL-MS) provides a wealth of information about the three-dimensional (3D) structure of proteins and their interactions. We introduce MaxLynx, a novel computational proteomics workflow for XL-MS integrated into the MaxQuant environment. It is applicable to noncleavable and MS-cleavable cross-linkers. For both, we have generalized the Andromeda peptide database search engine to efficiently identify cross-linked peptides. For noncleavable peptides, we implemented a novel dipeptide Andromeda score, which is the basis for a computationally efficient N-squared search engine. Additionally, partial scores summarize the evidence for the two constituents of the dipeptide individually. A posterior error probability (PEP) based on total and partial scores is used to control false discovery rates (FDRs). For MS-cleavable cross-linkers, a score of signature peaks is combined with the conventional Andromeda score on the cleavage products. The MaxQuant 3D peak detection was improved to ensure more accurate determination of the monoisotopic peak of isotope patterns for heavy molecules, which cross-linked peptides typically are. A wide selection of filtering parameters can replace the manual filtering of identifications, which is often necessary when using other pipelines. On benchmark data sets of synthetic peptides, MaxLynx outperforms all other tested software on data for both types of cross-linkers and on a proteome-wide data set of cross-linked Drosophila melanogaster cell lysate. The workflow also supports ion mobility-enhanced MS data. MaxLynx runs on Windows and Linux, contains an interactive viewer for displaying annotated cross-linked spectra, and is freely available at https://www.maxquant.org/.

Highlights

Chemical cross-linking combined with mass spectrometry (XLMS) has undergone remarkable developments to become a promising complementary method for studying protein structure, conformation, and interactions.[1−4] A typical protein cross-linking experiment starts with a formation of covalent bonds between spatially close residues in proteins or protein complexes through a cross-linker
The novel peak refinement feature (Figure 1b) is executed after the peak detection for data without ion mobility and assembles peaks that were not properly put together due to noise, which may happen for peptides with higher masses
3D peaks are first assembled into putative clusters by assembling peaks at the same m/z value and that are separated in retention time, whenever there is a third 3D peak in an m/z distance that corresponds to a neighboring peak in an isotope pattern that would cover the gap in retention time

Summary

Introduction

Chemical cross-linking combined with mass spectrometry (XLMS) has undergone remarkable developments to become a promising complementary method for studying protein structure, conformation, and interactions.[1−4] A typical protein cross-linking experiment starts with a formation of covalent bonds between spatially close residues in proteins or protein complexes through a cross-linker. The peptide mixture is analyzed using liquid chromatography coupled to tandem mass spectrometry (LC−MS/MS), and the resulting experimental MS/MS spectra are assigned to cross-linked peptides by using specialized algorithms.[4,9] Eventually, the cross-linked peptide identifications are used to gain insights into protein structures or their interaction partners.[10,11]

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