Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Similarities and differences in the structures and proteoform profiles of the Complement proteins C6 and C7

ABSTRACT: In this study, we used an integrative structural MS-based approach combining native MS, glycopeptide-centric MS, in-gel cross-linking MS (IGX-MS) to describe structural and glycosylation features of soluble C7. We compare this data with structural and glycosylation data for C6. For native MS analysis, proteins were buffer-exchanged into 150 mM aqueous ammonium acetate (pH 7.5) and analyzed on a modified Q-Exactive Plus Orbitrap mass spectrometer with extended mass range (EMR). For glyco-peptide centric analysis LC-MS and MS/MS analysis, C6 and C7 were digested by trypsin and a combination of Glu-C and trypsin. The resulting peptide mixtures were desalted, dried, and reconstituted in formic acid before MS analysis. All peptides were separated and analyzed using an Agilent 1290 Infinity HPLC system coupled on-line to an Orbitrap Q Exactive HF mass spectrometer. For the cross-linking analysis C7 gel band was cut from a BN-PAGE and cross-linked in-gel. The cross-linked protein were digested in the gel and data was acquired using an Ultimate 3000 system coupled on-line to an Orbitrap Exploris. The raw native MS spectra were deconvoluted to zero-charge spectra by Intact Mass software (Protein Metrics, CA, USA). The spectra were annotated using an in-house developed R script for semi-automated peak annotation, as described previously. The glycopeptide-centric bottom-up raw data were processed by the software Byonic and Skyline. Cross-linking raw data was searched in Proteome discoverer using the XlinkX node.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive Plus, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Albert J.R. Heck

PROVIDER: MSV000087019 | MassIVE | Tue Mar 09 09:32:00 GMT 2021

REPOSITORIES: MassIVE

ACCESS DATA

Json Xml

Similar Datasets

"In-gel cross-linking mass spectrometry (IGX-MS) of proteins and protein complexes"

Project description:Cross-linking mass spectrometry has developed into an important method to study protein structures and interactions. The in-solution cross-linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross-links obtained from co-occurring protein oligomers, complexes, or conformers. Here we developed a cross-linking workflow combining blue native PAGE with in-gel cross-linking mass spectrometry (IGX-MS). This workflow circumvents steps, such as buffer exchange and cross-linker concentration optimization. Additionally, IGX-MS enables the parallel analysis of co-occurring protein complexes using only small amounts of sample. Another benefit of IGX-MS observed by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of artificial over-length cross-links when compared to in-solution cross-linking. We next used IGX-MS to investigate the complement components C5, C6, and their hetero-dimeric C5b6 complex. The obtained cross-links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX-MS can be used to provide new insights into the initial stages of the terminal complement pathway.

2020-12-14 | PXD020014 | Pride

Peptide centric detection of cross-linked peptides using PASEF, MeroX, and Skyline

Project description:Cross-linking mass spectrometry (XL-MS) has evolved into a pivotal technique for probing protein interactions, with recent ad-vancements significantly enhancing its analytical potential. This technical note presents advancements in cross-linking mass spec-trometry (XL-MS) through the implementation of Parallel Accumulation-Serial Fragmentation (PASEF) on timsTOF instruments, enhancing the detection and analysis of protein interactions. Addressing the challenges in XL-MS, such as the interpretation of complex spectra, low abundance of cross-linked peptides (XL-peptides), and data acquisition biases, the study integrates a peptide-centric approach for the analysis of XL-MS data and presents the foundation for integrating data independent acquisition (DIA) in XL-MS with a vendor neutral and open-source platform. A novel method is described for processing DDA-PASEF data using Bruker software, enabling the conversion of this data into a format compatible with MeroX and Skyline tools. This approach, demonstrated with a bovine serum albumin (BSA) case study, significantly improves the identification and understanding of protein interactions, equipping the cross-linking community to overcome current analytical limitations.

2024-05-13 | PXD047307 | panorama

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

Project description:Advancements in cross-linking mass spectrometry (XL-MS) for structural analysis of proteins bridge the gap between purified systems and native tissue environments. Here, isobaric quantitative protein interaction reporter technology (iqPIR) was utilized to further extend XL-MS to the first system-wide comparative study of mitochondrial proteins from healthy and diseased murine hearts. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition. Structural insight into ketone-oxidation metabolons, OXPHOS machinery, and nucleotide transporter hybrid-conformations support mitochondrial remodeling in failing hearts while bringing forth new hypotheses for pathological mechanisms. The application of quantitative cross-linking technology in tissue provides molecular-level insight into complex biological systems challenging to model in cell culture, thus providing a valuable resource for studying human diseases.

2023-02-27 | PXD035712 | Pride

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

2022-08-02 | PXD035622 | Pride

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

Project description:Advancements of cross-linking mass spectrometry (XL-MS) for structural analysis of proteins bridges the gap between purified systems and native tissue environments. Here, we utilize isobaric quantitative protein interaction reporter technology (iqPIR) to further extend XL-MS to the first system-wide comparative study of mitochondrial proteins from healthy and diseased murine hearts. The failing heart interactome includes 544 statistically significant cross-linked peptide pairs altered in disease condition. Structural insight into ketone oxidation metabolons, OXPHOS machinery, and nucleotide transporter hybrid-conformations, support mitochondrial remodeling in failing heart while bringing forth new hypotheses for pathological mechanisms. Application of quantitative cross-linking technology in live tissue provides molecular-level insight to complex biological systems difficult to model in cell culture, thus providing a valuable resource for study of human diseases.

2022-08-02 | PXD027757 | Pride

Drug-specific signatures of the glycopeptide antibiotics dalbavancin, vancomycin and chlorobiphenyl-vancomycin in Streptomyces coelicolor

Project description:We used transcriptome profiling by RNAseq to identify the gene expression signatures elucidated in S. coelicolor in response to the three different glycopeptide compounds that share high degree of structural similarities and the same primary mode of action: dalbavancin, vancomycin and chlorobiphenyl-vancomycin.

2021-02-15 | E-MTAB-9846 | biostudies-arrayexpress

Optimized cross-linking mass spectrometry for in situ interaction proteomics

Project description:ecent development of mass spectrometer cleavable protein cross-linkers and algorithms for their spectral identification now permits large-scale cross-linking mass spectrometry (XL-MS). Here, we optimized the use of cleavable disuccinimidyl sulfoxide (DSSO) cross-linker for labeling native protein complexes in live human cells. We applied a generalized linear mixture model to calibrate cross-link peptide-spectra matching (CSM) scores to control the sensitivity and specificity of large-scale XL-MS. Using specific CSM score thresholds to control the false discovery rate, we found that higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) can both be effective for large-scale XL-MS protein interaction mapping. We found that the density and coverage of protein-protein interaction maps can be significantly improved through the use of multiple proteases. In addition, the use of sample-specific search databases can be used to improve the specificity of cross-linked peptide spectral matching. Application of this approach to human chromatin labeled in live cells recapitulated known and revealed new protein interactions of nucleosomes and other chromatin-associated complexes in situ. This optimized approach for mapping native protein interactions should be useful for a wide range of biological problems.

2019-04-03 | PXD010796 | Pride

Mass spectrometry study of formaldehyde cross-linking of structured proteins

Project description:Formaldehyde is a widely used fixative in biology and medicine. The current chemical model for formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this model. Instead, our data show that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield unusual fragments with a modification of one carbon atom. We apply this new understanding of the underlying cross-linking chemistry to the structural approach of cross-linking coupled to mass spectrometry. First, we cross-linked a mixture of purified proteins with formaldehyde. Our new analysis readily identified tens of cross-links from these proteins, which fit well with their atomic structures. We then perform in-situ cross-linking of human cells in culture. We identified 469 intra-protein and 90 inter-protein cross-links, which also fit well with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. We highlight an example in which formaldehyde cross-links localize the binding site of βNAC on the ribosome. We also find several interactions of actin with auxiliary proteins. Our findings not only expand our understanding of formaldehyde reactivity and toxicity, but also clearly demonstrate how to use this potent reagent for structural studies.

2020-06-25 | PXD015435 | Pride

GNPS - Systematic analysis of NDUFAF6 in complex I assembly and mitochondrial disease

Project description:Cross-linking AE-MS dataset for NDUFAF6 interactions

2024-03-08 | MSV000094276 | GNPS

Glycopeptides from recombinant EBV gH, gL and gp42 with EThcD

Project description:Soluble ectodomains of Epstein Barr Virus glycoproteins gH/gL and gp42 were expressed in HEK293F cells for structural characterization by cryo electron microscopy. The purified material was additionally analyzed by LC-MS/MS after digestion with trypsin or chymotrypsin to identify N-linked glycosylation from EThcD glycopeptide fragmentation spectra.

2018-04-10 | PXD006403 | 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