Abstract
Cross-linking/mass spectrometry resolves protein–protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labeled or collision-induced dissociation (CID)-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior of 910 unique cross-linked peptides in high-resolution MS1 and MS2 from published data and validate the observation by a ninefold larger set from currently unpublished data to explore if detailed understanding of their fragmentation behavior would allow computational delivery of information that otherwise would be obtained via isotope labels or CID cleavage of cross-linkers. Isotope-labeled cross-linkers reveal cross-linked and linear fragments in fragmentation spectra. We show that fragment mass and charge alone provide this information, alleviating the need for isotope-labeling for this purpose. Isotope-labeled cross-linkers also indicate cross-linker-containing, albeit not specifically cross-linked, peptides in MS1. We observed that acquisition can be guided to better than twofold enrich cross-linked peptides with minimal losses based on peptide mass and charge alone. By help of CID-cleavable cross-linkers, individual spectra with only linear fragments can be recorded for each peptide in a cross-link. We show that cross-linked fragments of ordinary cross-linked peptides can be linearized computationally and that a simplified subspectrum can be extracted that is enriched in information on one of the two linked peptides. This allows identifying candidates for this peptide in a simplified database search as we propose in a search strategy here. We conclude that the specific behavior of cross-linked peptides in mass spectrometers can be exploited to relax the requirements on cross-linkers.
Highlights
From the ‡Department of Bioanalytics, Institute of Biotechnology, Technische Universitat Berlin, 13355 Berlin, Germany; §Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, United Kingdom
Cross-linking/mass spectrometry extends the use of massspectrometry-based proteomics from identification [1, 2], quantification [3], and characterization of protein complexes [4] into resolving protein structures and protein–protein interactions [5,6,7,8]
Evaluation of the Predictive Power to Distinguish Linear and CrossLinked Fragments—Based on the ground truth of 910 peptide-spectrum match (PSM), we evaluated the predictive power of the relative fragment mass and the charge state as indicators whether or not a fragment is cross-linker containing
Summary
Spectra Collection and Filtering—We collected database search results from experiments that were acquired and described in previous publications [31,32,33] (Pride: PXD002142, PXD001835, PXD001454) and accumulated cross-linked and linear peptide spectrum matches (PSMs). Data Extraction—Software written in Python (2.7, www.python.org) was used to extract relevant fragmentation information from the local PostgreSQL database containing details about search settings and spectra annotations. The respective intensity rank for each matched fragment was stored. We compared b- or y-ion intensities for fragments in relation to the linker position or the peptide length, disregarding the specific ion index information (e.g. y7). Similarity Computation of Linear and Cross-Linked Spectra—The similarity comparison of two spectra was realized via an adapted ranked dot product scoring scheme.
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