Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Exploring Alternative Cysteine-reactive Chemistry for MS-cleavable Cross-linking Mass Spectrometry

ABSTRACT: To enable global PPI mapping, we have explored an alternative cysteine labeling chemistry, and thus designed and synthesized a sulfoxide-containing MS-cleavable haloacetamide-based cross-linker, DiBromoAcetamide SulfOxide (DBrASO). In combination with a newly developed SEC-HpHt fractionation method, we have successfully applied DBrASO for cross-linking of HEK293 cell lysates and demonstrated its capability to complement lysine-reactive reagents in order to expand PPI coverage at systems-level.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Fenglong Jiao

LAB HEAD: Lan Huang

PROVIDER: PXD037443 | Pride | 2023-07-20

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	OrbitrapLUMOS_20201112_01.raw	Raw
	OrbitrapLUMOS_20201112_03.raw	Raw
	OrbitrapLUMOS_20201112_05.raw	Raw
	OrbitrapLUMOS_20201112_07.raw	Raw
	OrbitrapLUMOS_20201112_09.raw	Raw

Items per page:

1 - 5 of 74

Publications

Exploring an Alternative Cysteine-Reactive Chemistry to Enable Proteome-Wide PPI Analysis by Cross-Linking Mass Spectrometry.

Jiao Fenglong F Salituro Leah J LJ Yu Clinton C Gutierrez Craig B CB Rychnovsky Scott D SD Huang Lan L

Analytical chemistry 20230118 4

The development of MS-cleavable cross-linking mass spectrometry (XL-MS) has enabled the effective capture and identification of endogenous protein-protein interactions (PPIs) and their residue contacts at the global scale without cell engineering. So far, only lysine-reactive cross-linkers have been successfully applied for proteome-wide PPI profiling. However, lysine cross-linkers alone cannot uncover the complete PPI map in cells. Previously, we have developed a maleimide-based cysteine-reacti ...[more]

PMID: 36652389

Similar Datasets

Enabling Photoactivated Cross-linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-cleavable Cross-linkers

Project description:Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions non-targetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions due to nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MSn. The MSn-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.

2021-05-07 | PXD022690 | Pride

DSBSO-based XL-MS Analysis of Breast Cancer PDX Tissues to Delineate Protein Interaction Network in Clinical Samples

Project description:We applied our previously established Alkyne-A-DSBSO-based in vivo XL-MS analytical platform on two types of breast cancer PDX samples cross-linking. Coupled with the LC-MSn strategy, the experiments enable us to obtain information on cancer-related protein-protein interactions since experimenting on PDX models.

2024-03-20 | PXD046966 | Pride

Integrative Structural Characterization of the Human Canonical and Non-canonical Cop9 Signalosome Complexes

Project description:The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that functions as a deneddylase to inactivate Cullin-RING E3 ligases for controlling protein ubiquitination. CSN possesses structural flexibility that is important for its activation upon binding to a diverse array of CRLs. The canonical and non-canonical CSN complexes consist of 8 (CSN1-8) and 9 (CSN1-9) subunits, respectively. Although CSN9 is not essential for CSN assembly and function, it appears to be important in non-catalytic regulation of CRLs by CSN. Here we employed a combinatory cross-linking mass spectrometry (XL-MS) approach to generate the largest PPI maps of human CSN complexes, which significantly enhanced the precision of integrative structural modeling. The resulting integrative structures allowed us not only to elucidate architectures of both complexes, but also to assess CSN structural dynamics that was not described in the crystal structure. In addition, we have determined CSN9 docking sites and its impact on the CSN structure. While CSN9 binding did not induce global conformational changes, it triggered subunit local reorientations that may be associated with CSN9 involvement in CSN-mediated steric regulation of CRLs.

2020-02-04 | PXD014673 | 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

Developing In Vivo Cross-linking Mass Spectrometry (XL-MS) to Decipher Protein Interaction Landscapes at the Proteome Scale

Project description:We have developed a new and robust in vivo cross-linking mass spectrometry (XL-MS) that utilizes a multifunctional MS-cleavable cross-linker to enable the efficient capture, enrichment, and identification of in vivo cross-linked peptides at the whole proteome scale.

2021-07-30 | PXD012788 | Pride

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

Mango-EcoliBDP - Mango: A General Tool for Collision Induced Dissociation-Cleavable Cross-Linked Peptide Identification

Project description:Dataset presented in Mango manuscript. Contains Ecoli cross-linked with BDP-NHP. Demonstration of whole proteome in-vivo cross-linking without an instrument capable of serial fragmentation.

2019-02-25 | PXD008975 | Pride

NefCD4AP2 Complex Crosslinking with DSSO for Integrative Structure Determination

Project description:Objective: HIV-1 Nef suppresses immune surveillance mechanisms to promote viral pathogenesis. Cellular receptor CD4 is essential for HIV entry into host cells, though becomes problematic at later stages in the virus replication cycle. CD4 at the plasma membrane is targeted by Nef for endocytosis and lysosomal degradation via recruitment of clathrin adaptor complex 2 (AP2 complex). Our goal is to use cross-linking mass spectrometry and integrative modeling to aid in structure determination of flexible portions of the Nef-CD4-AP2 complex. Methods: We use disuccinimidyl sulfoxide (DSSO), a MS-cleavable, bifunctional amine-reactive small molecule, to cross-link proximal Lys residues or N-termini of a purified reconstituted Nef-CD4 C-terminal domain fusion protein bound to the AP2Δμ2-CTD complex. Cross-linked proteins were separated by SDS-PAGE, excised from the gel and digested with trypsin. The resulting peptides were analyzed by specialized LC-MS3 experiments for identification of cross-linked residues using MSconvert (to obtain individual MS2 and MS3 level mgf files), ProteinProspector (to obtain peptide identification results files), and XLTools (to identify cross-linked peptides from results, MS2, and MS3 files). Additional scripts were used to summarize cross-linked results. Results: We find that an intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. A pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef’s activities and sensitize the virus to immune clearance.

2021-09-09 | PXD019338 | Pride

2) Twisting Urea- to Imide-Based Mass Spectrometry-Cleavable Cross-Linkers to Embed Affinity Tags

Project description:Disuccinimidyl dibutyric urea (DSBU) is a mass spectrometry (MS)-cleavable cross-linker that has been applied to multiple applications in structural biology, ranging from isolated protein complexes to comprehensive system-wide interactomics. DSBU facilitates a rapid and reliable identification of cross-links through the dissociation of its urea group in the gas-phase. In this study, we further advance the structural capabilities of DSBU by twisting the urea group into an imide, thus introducing a novel class of cross-linkers. This modification preserves the MS-cleavability of the amide bond, granted by the two acyl groups of the imide function. The central nitrogen atom enables the introduction of affinity purification tags. Here, we introduce disuccinimidyl dibutyric imide (DSBI) as prototype of this class of cross-linkers. It features a phosphonate handle for immobilized metal ion affinity chromatography (IMAC) enrichment. We detail DSBI synthesis and describe its behavior in solution and in the gas-phase while cross-linking isolated proteins and human cell lysates. DSBI and DSBU cross-links are compared at the same enrichment depths to bridge these two cross-linker classes. We validate DSBI cross-links by mapping them in high-resolution structures of large protein assemblies . The cross-links observed yield insights into the morphology of intrinsically disordered proteins (IDPs) and their complexes. The DSBI linker might spearhead a novel class of MS-cleavable and enrichable cross-linkers

2024-09-03 | PXD050960 | Pride

Quantitative cross-linking/mass spectrometry

Project description:Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue- resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. Wefound two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible. Biological significance Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue- resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so.We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Ourwork lays the foundations of studying themolecular details of biological processes at greater ease than this could be done so far.

2015-06-08 | PXD002142 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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