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

Abstract

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.

Cell fixation improves performance ofin situcrosslinking mass spectrometry while preserving cellular ultrastructure

“…After crosslink installation, cells are lysed and digested in a standard "bottom-up" workflow common to conventional proteomics routines, generating linked peptides for detection by MS. Crosslinked peptides are minor reaction products and cannot be identified with the conventional database searching strategies used in the proteomics community. New software makes the detection of crosslinks more efficient than ever before [14][15][16][17][18] , but very little progress has been made in improving the crosslinking reaction itself.…”