MassIVE MSV000099919

Complete Public PXD070779

Identification of Transglutaminase 2 associated proteins in HUVEC

Subscribe Comment Convert Spectra Reanalyze Spectra Compare Results Add Reanalysis

Description

TG2 is physiologically expressed in HUVEC cells, contributing to the maintenance of cardiovascular homeostasis, but the mechanistic details remain unclear. Identification of TG2-associated proteins can provide a deeper view of TG2's role in endothelial cells. We have applied two approaches: First, a site-specifically N-terminally biotinylated recombinant TG2 was produced in the Rosetta 2 strain, and the Neutravidin-agarose resin was used to isolate TG2-associated proteins from the immortalised HUVEC cell extract. Second, we have developed an endogenous TG2-silenced, N-terminally triple-Flag-tagged transgenic TG2-expressing HUVEC cell line, and intracellularly assembled TG2-associated protein complexes were isolated using anti-Flag agarose resin. To reveal the conformation-dependent interactions of TG2, NC9 (24 hours treatment before lysis) and GTPgammaS (during CoIP) were applied to stabilise open or closed TG2, respectively. (One acidic and a denaturing elution were applied in the case of each sample from the appropriate resin. The eluted sample was run into an SDS-polyacrylamide gel till the top of the separation gel, and the protein-containing part of the gel was cut based on Coomassie (Page-Blue) staining. The proteins in the submitted gel pieces were digested with trypsin overnight (37 C degree). After digestion, the samples were dried to a volume of 0.01 ml. The protein samples were prepared for MS analysis using the Easy nLC1200 (Thermo Scientific) nanouplc-Orbitrap Fusion (Thermo Scientific) MS/MS system. The results were imported into the Scaffold 5.0.1 software. In the Scaffold software, the Exclusive Unique Peptide Count display option and a protein threshold of 1.0% false discovery rate (FDR), along with a peptide threshold of 0.1% FDR, were applied, requiring a minimum of one identified peptide for each protein. The fragmentation table of each detected unique peptide was meticulously checked to verify that the detected peptide is a genuine hit. For this verification, the fragmentation table must include at least four B or Y ion series. The experiments were repeated at least 3 times, and the hits obtained from each repetition were aggregated, after which the control non-specifically bound protein hits were deducted.) [doi:10.25345/C5B853X4J] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: transglutaminase 2 ; HUVEC ; CoIP ; protein-protein interaction ; conformation dependence ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		Robert Kiraly, University of Debrecen, Hungary
Submitting User: kiralyr
Number of Files:
Total Size:
Spectra:
Subscribers:
 
Owner Reanalyses
Experimental Design
    Conditions:
    Biological Replicates:
    Technical Replicates:
 
Identification Results
    Proteins (Human, Remapped):
    Proteins (Reported):
    Peptides:
    Variant Peptides:
    PSMs:
 
Quantification Results
    Differential Proteins:
    Quantified Proteins:
 
Browse Dataset Files Browse Results
 
FTP Download Link (click to copy):

Species

Instrument

Modifications

- Dataset Reanalyses

+ Dataset History

Click here to queue conversion of this dataset's submitted spectrum files to open formats (e.g. mzML). This process may take some time.

When complete, the converted files will be available in the "ccms_peak" subdirectory of the dataset's FTP space (accessible via the "FTP Download" link to the right).
Number of distinct conditions across all analyses (original submission and reanalyses) associated with this dataset.

Distinct condition labels are counted across all files submitted in the "Metadata" category having a "Condition" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct biological replicates across all analyses (original submission and reanalyses) associated with this dataset.

Distinct replicate labels are counted across all files submitted in the "Metadata" category having a "BioReplicate" or "Replicate" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct technical replicates across all analyses (original submission and reanalyses) associated with this dataset.

The technical replicate count is defined as the maximum number of times any one distinct combination of condition and biological replicate was analyzed across all files submitted in the "Metadata" category. In the case of fractionated experiments, only the first fraction is considered.

"N/A" means no results of this type were submitted.
Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

"N/A" means no results of this type were submitted.
Number of distinct protein accessions reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct unmodified peptide sequences reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct peptide sequences (including modified variants or peptidoforms) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Total number of peptide-spectrum matches (i.e. spectrum identifications) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins quantified across all analyses (original submission and reanalyses) associated with this dataset.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

A protein is differentially abundant if its change in abundance across conditions is found to be statistically significant with an adjusted p-value <= 0.05 and lists no issues associated with statistical tests for differential abundance.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
This dataset may not contain all raw spectra data as originally deposited in PRIDE. It has been imported to MassIVE for reanalysis purposes, so its spectra data here may consist solely of processed peak lists suitable for reanalysis with most software.