MassIVE MSV000094031

Partial Public PXD049247

LC-MS/MS based proteomics analysis of decellularized and non-decellularized mouse kidney tissue samples

Subscribe Comment Convert Spectra Add Reanalysis

Description

Two C57Bl6/J male mice were sacrificed by injecting 200 microliters of the lethal anaesthetic Dolethal. Kidneys were perfused with Dulbeccos Phosphate Buffered Saline, dissected, decapsulated and weighted. Kidneys were decellularized using 3 freeze-thaw cycles (freezing at -80C, 30 minute long thawing at 4C) and a 3-day long incubation in 2% SDS at 4C under gentle rotation. Ten to twenty milligram tissue net weight of each sample was homogenised. Protein concentration was determined using Bradford Assay. Ten micrograms of total protein was loaded in SDS-PAGE and migrated for 25min and prepared using the GeLC-MS method (doi: 10.1007/7651_2017_76). For LC-MS/MS analysis a Dionex Ultimate 3000 HPLC nanoflow system was used in combination with a Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer. Samples were reconstituted in 25 microliters mobile phase A (98% water, 2% acetonitrile, 0.1% formic acid) and 10 microliters were loaded onto the LC column configured with a Dionex 0.1 x 20 mm, 5 micrometres, 100 A C18 nano trap column with a 5 microliters/min flow rate. An Acclaim PepMapTM C18 100 nanoViper column 75 micrometres x 50cm, 2 micrometres 100 A was used with a flow rate of 300nL/min. Peptides were eluted under a 250min gradient from 2% mobile phase B (0.1% formic acid in 80% acetonitrile) to 95% mobile phase B. Voltage of the positive ion electrospray ionization was set to 2.1kV. The mass spectrometer parameters were set as follows: 1) full MS: resolution of 70,000, automatic gain control (AGC) target at 1e6, maximum injection time (IT) of 100ms, scan range of 380 - 1,200m/z; 2) dd-MS2: resolution of 17,500, AGC target at 1e5, maximum IT of 50ms, TopN of 20, normalized collision energy of 30; 3) dd settings: intensity threshold 1.6e5, Minimum AGC target 8.00e3, dynamic exclusion for 15s. Raw files were processed with Thermo Proteome Discoverer 2.4 software, utilizing the Sequest search engine and the UniProt Mus musculus fasta database containing only canonical sequences (downloaded on 31/05/2023, including 17,155 reviewed entries). Carbamidomethylation of cysteine was set as a static modification, and oxidation of methionine and oxidation of proline were set as dynamic modifications. Two missed cleavage sites, a precursor mass tolerance of 5ppm, and fragment mass tolerance of 0.05Da were allowed. Feature mapper node was run with default settings. [doi:10.25345/C5D50G80B] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: LC-MS/MS ; proteomics ; ECM ; mouse ; kidney ; decellularization

Contact

Principal Investigators:
(in alphabetical order) 		Antonia Vlahou, Biomedical Research Foundation, Academy of Athens, Greece
Jean-Sebastien Saulnier-Blache, Institut National de la Sante et de la Recherche Medicale, U1048, Institute of Cardiovascular and Metabolic Disease, Toulouse and Universite Toulouse III Paul-Sabatier, Toulouse, France
Submitting User: mmakrid

Publications

Frattini T, Devos H, Makridakis M, Roubelakis MG, Latosinska A, Mischak H, Schanstra JP, Vlahou A, Saulnier-Blache JS.
Benefits and limits of decellularization on mass-spectrometry-based extracellular matrix proteome analysis of mouse kidney.
Proteomics.2024 Sep;24(17):e2400052. doi: 10.1002/pmic.202400052.

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
 
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.