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Retention time as an extra dimension for proteome-wide cross-link identification

ABSTRACT: Chemical crosslinking methods have not been successfully applied to protein-protein interaction discovery mainly due to the computation complexity (O (n2)) issue. In a previous report, we proposed a decision tree searching strategy which can reduce complexity by orders of magnitude. In this study, we found that the mono-linked peptides carry out the information of the retention time of the cross-linked pairs, therefore the retention time of cross-linked peptide pairs can be predicted accurately. By utilizing the obtained retention time information, the false positive rate can be reduced by around 86% with a sensitivity loss of 12%. The method was successfully applied to the identification of inter cross-links obtained from E. coli cell lysate crosslinked by a newly synthesized enrichable crosslinker.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Bos Taurus (bovine) Escherichia Coli

TISSUE(S): Blood Plasma, Cell Culture

SUBMITTER: Hongli Chen

LAB HEAD: Hongli Chen

PROVIDER: PXD024253 | Pride | 2021-07-29

REPOSITORIES: Pride

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			Action	DRS
	117-pDSBE-BSA.pdStudy	Other
	117-pDSBE-BSA.pdStudy.bak	Other
	117-pDSBE-BSA2-_1_.msf	Msf
	117-pDSBE-BSA2-_1_.msfView	Msf
	117-pDSBE-BSA2-_1_.mzML	Mzml

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Retention time as an extra dimension for proteome-wide cross-link identification

Project description:Chemical crosslinking methods have not been successfully applied to protein-protein interaction discovery mainly due to the computation complexity (O (n2)) issue. In a previous report, we proposed a decision tree searching strategy which can reduce complexity by orders of magnitude. In this study, we found that the mono-linked peptides carry out the information of the retention time of the cross-linked pairs, therefore the retention time of cross-linked peptide pairs can be predicted accurately. By utilizing the obtained retention time information, the false positive rate can be reduced by around 86% with a sensitivity loss of 12%. The method was successfully applied to the identification of inter cross-links obtained from E. coli cell lysate crosslinked by a newly synthesized enrichable crosslinker.

2021-08-06 | PXD027831 |

Cross-linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics

Project description:The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) Bovine serum albumin, (ii) E. coli ribosome, and (iii) HEK293T cell nuclear extracts. We identified a total of 623 unique cross-linking sites for BSA, 670 for the E. coli ribosome, and 1,617 unique cross-links for nuclear extracts, corresponding to 1,088 intra- and 529 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving 3D-structures of single proteins, but also for performing system-wide protein interaction studies.

2022-03-17 | PXD027234 | Pride

Analysis of bilirubin-induced bovine serum albumin stability using protein denaturation and quantitative cross-linking mass spectrometry

Project description:We report the combination of protein-denaturation stability principles with quantitative cross-linking mass spectrometry using isobaric quantitative protein interaction reporter technologies. This method enables the evaluation of ligand-induced protein engagement through analysis of cross-link relative ratios across chemical denaturation. We found ligand-stabilized cross-linked lysine pairs in bovine serum albumin (BSA) and ligand bilirubin map to known binding sites Sudlow Site I and subdomain IB.

2024-07-03 | PXD036649 | Pride

Transcriptome wide mapping of Hfq mediated RNA-RNA interactions in E. coli (RIL-seq)

Project description:Most E. coli sRNAs interact with their mRNA targets through simultaneous binding to the Hfq chaperon. In this experiment we cross-linked RNA to proteins in-vivo then did Hfq IP followed by ligation of bound RNAs and sequencing to identify sRNA-mRNA interactions. We termed the method RIL-seq for RNA Interactions by Ligation - sequencing.

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capsPASEF – Collisional Cross Section Assisted Precursor Selection Applied to Cross-linking Mass Spectrometry

Project description:Ion mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, HCD cell and a time-of-flight mass spectrometer, to interrogate ions at high speeds with on-the-fly fragmentation. Ion mobility is perfectly suited for cross-linking mass spectrometry, which aims to uncover spatial restraints for proteins. The used cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. This technique however suffers in terms of the low abundance of cross-linked peptides, which is partially resolved with enrichable cross-linking reagents. This class of reagents enables selective enrichment of cross-linking reagent modified peptides, resulting in selection of the cross-linked peptide pairs and peptides connected to a partially hydrolyzed reagent – termed mono-links. Even though mono-links carry limited structural information, for experiments aiming to uncover protein interactions in an unbiased manner they are unwanted byproducts. Gas-phase separation by IM has the potential to separate the two products and, indeed, we find separation between mono-links and cross-linked peptide pairs for PhoX cross-linked proteins. Moreover, an easy-to-interpret division at a CCS of 500 Å and a mono-isotopic mass of 2 kDa is present, which can be used for precursor selection. From our experiments on low complexity protein systems, sequencing of 50 - 70% of the mono-links is prevented, allowing the mass spectrometer the focus on sequencing the relevant cross-links. Application to a highly complex lysate focusing provides a 10-50 % increase in detected cross-links.

2020-07-22 | PXD018189 | Pride

Mitochondrial interactome quantitation reveals structural changes in metabolic machinery in failing murine heart

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2022-08-02 | PXD027757 | Pride

Structural Analysis of 70S Ribosomes by Cross-Linking/Mass Spectrometry Reveals Conformational Plasticity

Project description:We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS.

2020-08-13 | PXD018935 | Pride

In planta chemical crosslinking and mass spectrometry analysis of protein structure and interaction in Arabidopsis

Project description:Mass spectrometry analysis in combination with the site-specific chemical cross-linking has emerged as a powerful method in study of three-dimensional structure of protein complex and in mapping of protein-protein interactions (PPIs). Even though in vitro cross-linking experiments have been widely applied to investigate the specific interactions of a bait protein and its targets, the measurement of in vivo protein tertiary structure and PPIs has been problematic and strenuous due to the dynamic nature of the biological systems and a lower number of cross-linked peptides that can be isolated via MudPIT (Multidimensional Protein Identification Technology) for mass spectrometry analysis. Using Arabidopsis thaliana as a model multicellular eukaryotic organism, we have attempted to develop an improved in vivo chemical cross-linking and mass spectrometry (or IPXL-MS) workflow, which aims at optimizing the in vivo cross-linking conditions, establishing of a MudPIT procedure for enrichment of cross-linked peptides, and developing an integrated software program to identify the in planta chemical cross-linked peptides, by which three pairs of in vivo cross-linked peptides of high-confidence has been identified twice from two independent biological replicates. This work has demarked a beginning of alternative proteomic approach in study of in vivo protein tertiary structure and PPIs in higher plants. This in vivo cross-linking approach may be applied into other model multicellular organisms, such as mouse, for molecular systems biological research.

2016-06-28 | PXD003658 | Pride

Real-time library search assisted identification of cross-links increases resolution for structural modeling and interactome mapping

Project description:Cross-linking mass spectrometry (XL-MS) is a universal tool of molecular and structural biology for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification because a large proportion of measurement time is spent on MS2 acquisitions of mono-links. Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tert-butyl-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of full scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Furthermore, RTLS improves cross-link identification from unenriched samples and short gradients, which is beneficial in high-throughput approaches and when instrument time or sample amount is limited.

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A Simple Cross-Linking/Mass Spectrometry Workflow to Study System-Wide Protein Interactions in Drosophila Embryos

Project description:We provide a cross-linking/MS workflow that can be applied to complex systems. The software tool MeroX 2.0 can be used to identify cross-linked peptide on a proteome-wide level. We applied the workflow to extracts of Drosophila embryos and identified 5,129 unique cross-linked residue pairs in biological triplicates.

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