Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength

ABSTRACT: Cross-linked samples consist of monomeric SPD-5 which were incubated with either kinase dead or constitutively active PLK-1 plus ATP-MgCl2 in a 150mM KCl, 25mM HEPES, pH7.4 buffer for 2 hours at room temperature. Samples were then cross-linked using 8mM DMTMM for 45min and quenched with 50 mM ammonium bicarbonate for 15 min at RT shaking at 300rpm. Samples ran on a 16-20% SDS-page gel revealing a monomer band and a dimer band in both conditions using a Commassie based stain. Monomer bands were excised, then digested overnight with trypsin (Pierce), reduced with DTT and alkylated with iodoacetamide (Sigma-Aldrich). Samples were cleaned using solid-phase extraction with an Oasis HLB plate, then injected into an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Samples 1122415-1122420: de-phosphorylated Samples 1123298-1123300: phosphorylated

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Caenorhabditis Elegans (ncbitaxon:6239)

SUBMITTER: Jeffrey Woodruff

PROVIDER: MSV000093775 | MassIVE | Fri Jan 05 07:19:00 GMT 2024

REPOSITORIES: MassIVE

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

A central helical hairpin in SPD-5 enables centrosome strength and assembly

Project description:Cross-linked samples consist of middle region of SPD-5 (541-677) plus 18 amino acids at the N-terminus (MGSSHHHHHHENLYFQSN) which we term F1. Two F1 samples (7.6 uM) were incubated with either kinase dead or constitutively active PLK-1 plus ATP-MgCl2 in a 150 mM KCl, 25mM HEPES, pH7.4 buffer for 2 hours at room temperature. Samples were then cross-linked using 8mM DMTMM for 45min and quenched with 50 mM ammonium bicarbonate for 15 min at RT shaking at 300rpm. Samples ran on a 16-20% SDS-page gel revealing a monomer band and a dimer band in both conditions using a coomassie based stain. Monomer bands (LUM2_1076612, PLK-1 KD, LUM2_1076614, PLK-1 -CA) and dimer bands (LUM2_1076613, PLK-1 KD, LUM2_1076615, PLK-1 KD) were excised.

2024-02-28 | MSV000094191 | MassIVE

Multivalent Coiled-Coil Interactions as a Conserved Design Principle for Centrosome Assembly

Project description:Crosslinking reactions were prepared at room temperature with 1uM dephosphorylated SPD-5, 1uM Kinase Dead (KD) PLK-1 , 0.2 mM ATP, 10 mM MgCl2, 150mM KCl, 25mM HEPES, pH7.4 and 0.5mM DTT. Samples were incubated for 2 hr at room temperature followed by addition of 1.47uL of DMTMM (20mg/mL) for 45min at room temperature (shaking at 300 rpm). To quench the reaction, we added 2.56uL of Ammonium Bicarbonate (50mM) for 15min at room temperature (shaking at 300rpm).

2024-02-28 | MSV000094199 | MassIVE

Multivalent Coiled-Coil Interactions as a Conserved Design Principle for Centrosome Assembly

Project description:Crosslinking reactions were prepared at room temperature with 1uM dephosphorylated SPD-5, 1uM Constitutively Active (CA) PLK-1, 0.2 mM ATP, 10 mM MgCl2, 150mM KCl, 25mM HEPES, pH7.4 and 0.5mM DTT. Samples were incubated for 2 hr at room temperature followed by addition of 1.47 uL of DMTMM (20mg/mL) for 45min at room temperature (shaking at 300 rpm). To quench the reaction, we added 2.56uL of Ammonium Bicarbonate (50mM) for 15min at room temperature (shaking at 300rpm).

2024-02-28 | MSV000094198 | MassIVE

Phosphorylation sites that influences the RNA-binding activity of Papi

Project description:We conducted UV cross-linking immunoprecipitation (CLIP) with and without phosphatase inhibitor and compared the outcome. The topmost band on the western blots appeared more prominent with the inhibitor. The RNA-binding activity of the band was also stronger with the inhibitor. We isolated the band and subjected to LC-MS/MS to identify the phosphorylated residues.

2022-02-01 | PXD027395 | JPOST Repository

Cross-linked ammonia monooxygenase complex in BN-PAGE gel from Nitrososphaera viennensis

Project description:A band containing the ammonia monooxygenase complex from Nitrosospheara viennensis was cut from a Blue Native PAGE gel and cross linked using DSSO.

2023-02-13 | PXD034475 | Pride

Enhancing Antimicrobial Peptide Potency through Multivalent Presentation on Coiled-Coil Nanofibrils.

Project description:Antibiotic-resistant microbes have become a global health threat. New delivery systems that enhance the efficacy of antibiotics and/or overcome the resistances can help combat them. In this context, we present FF03, a fibril-forming α-helical coiled-coil peptide that functions as an efficient scaffold for the multivalent presentation of the weakly cationic antimicrobial peptide (AMP) IN4. The resulting IN4-decorated FF03 coiled-coil fibrils (FF03 + IN4) are nonhemolytic and noncytotoxic and show enhanced antimicrobial activity relative to unconjugated IN4 and standard antibiotics against several bacterial strains. Scanning electron microscopy analysis shows that FF03 + IN4 nanofibers disrupt methicillin-resistant Staphylococcus aureus membranes, indicating a surface-level mode of action. Furthermore, transmission electron microscopy and circular dichroism studies indicate that decoration of the FF03 scaffold with IN4 does not alter the secondary-structure propensity or fibril-forming properties of FF03. Thus, the approach reported herein provides a new peptidic scaffold for the multivalent presentation of AMPs to obtain nonhemolytic and noncytotoxic antimicrobial systems with improved efficacy relative to the unconjugated AMP analogues.

| S-EPMC7812673 | biostudies-literature

A2M bait region engineering - autoconjugation product cross-link identification and PRM quantification

Project description:In-gel digest of a cross-linked product band was used to identify an internal isopeptide cross-link formed in A2M TR K704 upon bait region cleavage by trypsin. PRM was used to quantify the thiol ester in native PAGE bands of A2M nEXT and EXT mutants.

2021-09-10 | PXD023651 | Pride

Multivalent Coiled-Coil Interactions as a Conserved Design Principle for Centrosome Assembly

Project description:Purified SPD-5 (in Storage Buffer; 25 mM HEPES, pH 7.4, 500 mM KCl, 0.1% CHAPS, 1% glycerol) was diluted in Assembly Buffer (25 mM HEPES, pH 7.4, 150 mM KCl) and incubated for 2 hr at 23 C. Final conditions for mass spectrometry analysis of multimeric species included 1.7 mM protein, 200 mM KCl and 2 hr incubation at 23 C. For DMTMM cross-linking, 8 mM 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (Sigma-Aldrich) was added to the protein samples and incubated at 23 C with shaking for 45 min. Reactions were quenched with 50 mM ammonium bicarbonate and incubated at 23 C for 15 min.

2024-02-28 | MSV000094194 | MassIVE

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

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

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