Dataset Information

Engineered chemotaxis core signaling units

ABSTRACT: Identificaiton of bacterial chemoreceptors by DSSO-based MS2-MS3 method.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Escherichia Coli

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Sheng Zhang

LAB HEAD: SHENG ZHANG

PROVIDER: PXD019599 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

Engineered chemotaxis core signaling units indicate a constrained kinase-off state.

Muok Alise R AR Chua Teck Khiang TK Srivastava Madhur M Yang Wen W Maschmann Zach Z Borbat Petr P PP Chong Jenna J Zhang Sheng S Freed Jack H JH Briegel Ariane A Crane Brian R BR

Science signaling 20201110 657

Bacterial chemoreceptors, the histidine kinase CheA, and the coupling protein CheW form transmembrane molecular arrays with remarkable sensing properties. The receptors inhibit or stimulate CheA kinase activity depending on the presence of attractants or repellants, respectively. We engineered chemoreceptor cytoplasmic regions to assume a trimer of receptor dimers configuration that formed well-defined complexes with CheA and CheW and promoted a CheA kinase-off state. These mimics of core signal ...[more]

PMID: 33172954

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Project description:Over the past few decades cross-linking mass spectrometry (XLMS) has become a powerful tool for identification of protein-protein interactions and for gaining insight into the structures of proteins in living cells, tissues, and organelles. The development of new crosslinkers, enrichment strategies and data acquisition methods led to the establishment of numerous new software tools specifically for the analysis and interpretation of cross-linking data. We previously published one of these tools called MS Annika, a cross-linking search engine which can accurately identify cross-linked peptides in MS2 spectra from a variety of different MS-cleavable crosslinkers. In this publication we present an updated MS Annika and a new search algorithm that additionally supports processing of data from MS2-MS3-based approaches and identification of peptides from MS3 spectra. In the new MS2-MS3 search algorithm, MS3 spectra are matched to their corresponding precursor doublet peak in the MS2 scan to identify the crosslink modification and the monoisotopic peptide mass. This information is then used to adjust the MS3 spectra for search with MS Amanda, our in-house developed peptide search engine, to identify the cross-linked peptides. Peptides that are identified in the MS2 scan and one or more of the associated product MS3 scans are re-scored with a novel scoring function to reflect the increased confidence. Finally, the 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 on five different datasets covering a variety of experimental approaches and compared it to XlinkX and MaXLinker, two other cross-linking search engines that support MS3 crosslink identification. Three of the datasets were benchmark datasets of synthetic peptides that allow calculation of an experimentally validated FDR, and we show that MS Annika detects up to 4 times more true unique crosslinks than MaXLinker and up to 35% more than XlinkX while simultaneously yielding less false positive hits and therefore a more accurate FDR than the other two search engines. Additionally, for the other two datasets we could show that MS Annika finds between 74% to 2.5 times more crosslinks at 1% estimated FDR and reveals protein-protein interactions that are not detected by either XlinkX or MaXLinker.

Dataset Information

Engineered chemotaxis core signaling units

Publications

Engineered chemotaxis core signaling units indicate a constrained kinase-off state.

Similar Datasets

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