Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Optimized TMT-based quantitative cross-linking mass spectrometry strategy for large-scale interactomic studies

ABSTRACT: Cross-linking mass spectrometry is a powerful method for the investigation of protein-protein interactions from highly complex samples. XL-MS combined with tandem mass tag labeling holds the promise of large-scale PPI quantification. However, a robust and efficient TMT-based XL-MS quantification method has not yet been established due to the lack of a benchmarking dataset and thorough evaluation of various MS parameters. To tackle these limitations, we generate a two-interactome dataset by spiking-in TMT-labeled cross-linked E. coli lysate into TMT-labeled cross-linked HEK293T lysate using a defined mixing scheme. Using this benchmarking dataset, we assess the efficacy of cross-link identification and accuracy of cross-link quantification using different MS acquisition strategies. For identification, we compare various MS2- and MS3-based XL-MS methods, and optimize stepped HCD energies for TMT-labeled cross-links. We observed a need for notably higher fragmentation energies compared to unlabeled cross-links. For quantification, we assess the quantification accuracy and dispersion of MS2-, MS3- and synchronous precursor selection-MS3-based methods. We show that a stepped HCD-MS2 method with stepped collision energies 36-42-48 provides a vast number of quantifiable cross-links with high quantification accuracy. This widely applicable method paves the way for multiplexed quantitative PPI characterization from complex biological systems.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Escherichia Coli Bacteria

TISSUE(S): Cell Suspension Culture, Permanent Cell Line Cell, Cell Culture

SUBMITTER: Max Ruwolt

LAB HEAD: Fan Liu

PROVIDER: PXD031114 | Pride | 2022-04-07

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Optimized fragmentation improves the identification of peptides cross-linked using MS-cleavable reagents

Project description:Cross-linking mass spectrometry (XLMS) is becoming increasingly popular, and current advances are widening the applicability of the technique so that it can be utilized by non-specialist laboratories. Specifically, the use of novel mass spectrometry-cleavable (MS-cleavable) reagents dramatically reduces complexity of the data by providing i) characteristic reporter ions and ii) the mass of the individual peptides, rather than that of the cross-linked moiety. However, optimum acquisition strategies to obtain the best quality data for such cross-linkers with higher energy C-trap dissociation (HCD) alone is yet to be achieved. Therefore, we have carefully investigated and optimized MS parameters to facilitate the identification of disuccinimidyl sulfoxide (DSSO)-based cross-links on HCD-equipped mass spectrometers. From the comparison of 9 different fragmentation energies we chose several stepped- HCD fragmentation methods that were evaluated on a variety of cross-linked proteins. The optimal stepped-HCD method was then directly compared with previously described methods using an Orbitrap Fusion™ Lumos™ Tribrid™ instrument using a high-complexity sample. The final results indicate that our stepped-HCD method is able to identify more cross-links than other methods, mitigating the need for multistage MS (MSn) enabled instrumentation and alternative dissociation techniques.

2019-02-01 | PXD011861 | Pride

The structures of R124H and R555W TGFBIp suggest a direct role of the mutated residues in the aggregation of TGFBIp in granular corneal dystrophy

Project description:Protein aggregation in the outermost layers of the cornea, leading to cloudy vision and in severe cases blindness, is linked to mutations in the extracellular matrix protein, transforming growth factor-β-induced protein (TGFBIp). Among the most frequent disease-causing mutations are R124H and R555W, both associated with granular corneal dystrophy (GCD) characterized by early-onset amorphous aggregation. The molecular mechanisms of protein aggregation in GCD are largely unknown. In this study, we determined the crystal structures of R124H and R555W. Whereas no structural changes of mutated monomeric TGFBIp can explain its aggregation propensity, the two mutations facilitate a new dimer interface in the crystal packing, not appearing in the wild-type structure. The interface in both R124H and R555W structures are centered on residue 124 in one TGFBIp molecule and 555 in a second TGFBIp molecule. This unique interface of R124H and R555W is not compatible with arginines at positions 124 and 555 as seen in wild-type TGFBIp. Hence, this intermolecular interaction may underlie the similar aggregation behavior exhibited by the two TGFBIp mutants in vivo. Using cross-linking mass spectrometry, we identified and characterized a dimer of TGFBIp wild-type and mutants in solution. The soluble dimers also involve interactions between the N- and C-terminal domains of two TGFBIp molecules but is not identical to the crystal packing dimer interface involving R124H and R555W. Targeting of the crystal packing dimer interface might offer new opportunities for therapeutic intervention to treat patients with GCD.

2021-09-08 | PXD022976 | Pride

Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions

Project description:Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions. The dMP is a robust 2-step protocol that ensures 95 % of irreversible denaturation within only 5 min. We show that dMP provides accurate mass identification across a broad mass range (30 kDa-5 MDa) along with direct label-free relative quantification of all coexisting XL species (sub-complexes and aggregates). We compare dMP with SDS-PAGE and observe that, unlike the benchmark, dMP is time-efficient (3 min/triplicate), requires significantly less material (20-100x) and affords single molecule sensitivity. To illustrate its utility for routine structural biology applications, we show that dMP affords screening of 20 XL conditions in 1 h, accurately identifying and quantifying all coexisting species. Taken together, we anticipate that dMP will have an impact on ability to structurally characterize more PPIs and macromolecular assemblies, expected final complexes but also sub-complexes that form en route.

2024-03-15 | PXD042549 | Pride

A2M-3K DSSO cross-linking to identify the bait region position

Project description:An A2M mutant, A2M-3K, with the bait region arginines replaced with lysines, was cross-linked with DSSO. Its monomeric gel band was analyzed by XL-MS to identify the bait region position within a subunit.

2021-05-21 | PXD019048 | Pride

Expanding the depth and sensitivity of cross-link identification by differential ion mobility using FAIMS

Project description:In cross-linking mass spectrometry (XL-MS), the depth and sensitivity of cross-link detection is often limited by the low abundance of cross-links compared to non-cross-linked peptides in the digestion mixture. To improve the identification efficiency of cross-links, here we present a gas-phase separation strategy using high field asymmetric waveform ion mobility spectrometry (FAIMS) coupled to the Orbitrap Tribrid mass spectrometers. By enabling an additional peptide separation step in gas phase using the FAIMS device, we increase the number of cross-link identification by 22% for a medium complex sample and 59% for strong cation exchange-fractionated HEK293T cell lysate in XL-MS experiments using disuccinimidyl sulfoxide (DSSO) cross-linker. When disuccinimidyl suberate (DSS) cross-linker is in use, we are able to boost cross-link identification by 89% for the medium and 100% for the highly complex sample comparing to the analyses without FAIMS . Furthermore, we show that for medium complex samples, FAIMS enables the collection of single-shot XL-MS data with comparable depth to the corresponding sample fractionated by chromatography-based approaches. Altogether, we demonstrate FAIMS is highly beneficial for XL-MS studies by expanding the proteome coverage of cross-links while improving the efficiency and confidence of cross-link identification.

2020-07-22 | PXD019926 | 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.

2023-03-20 | PXD037652 | Pride

CX-MS of DSSO-cross-linked A2M - Structural investigations of human α2-macroglobulin elucidate its native conformation and inhibitory mechanism

Project description:Human A2M was cross-linked with DSSO in its native, methylamine-treated, and trypsin-cleaved conformations. The monomer, dimer, and tetramer SDS-PAGE gel bands of native A2M were analyzed by in-gel digest. Total protein digests of all three conformations were analyzed and compared.

2021-05-21 | PXD019101 | Pride

HAP40 orchestrates huntingtin structure for differential interaction with polyglutamine expanded exon 1

Project description:Huntington’s disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo and in cell models that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass-spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The exon 1 region of HTT is dynamic but shows greater conformational variety in the polyglutamine expanded mutant than wildtype exon 1. Our data provide a foundation for future functional and drug discovery studies targeting Huntington’s disease and illuminate the structural consequences of HTT polyglutamine expansion.

2022-02-21 | PXD028313 | Pride

HAP40 orchestrates huntingtin structure for differential interaction with polyglutamine expanded exon 1

2022-09-09 | PXD028415 | Pride

Structural and functional analyses identify unique features of the Smc5/6 complex among SMC family of complexes

Project description:Cross-linking mass spectrometry data for integrative structural analyses of the Smc5/6 holo-complex from budding yeast. Electron microscopy, crosslinking mass spectrometry, and computational modeling reveal that while the complex shares an overall organization with other SMC complexes, it possesses several strikingly different features.

2021-05-05 | PXD023164 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data