MassIVE MSV000092097

Partial Public

Top-down proteomics of mouse beta cell carboxypeptidase E deletion reveals novel substrates and defects in hormone processing

Subscribe Comment Convert Spectra Reanalyze Spectra Add Reanalysis

Description

100 islets from wildtype and islet beta-cell Ins1Cre/+ ; Cpefl/fl mice were pooled and processed for top-down proteomic analysis. Briefly, homogenization buffer (8 M urea, 100 mM ammonium bicarbonate [ABC], 5 mM ethylenediaminetetraacetic acid [EDTA], 1 mM phenylmethylsulfonyl fluoride [PMSF]) was added to each sample before vortexing for 10 sec to resuspend islets followed by sonication in a water bath for 5 min at room temperature (rt). Reduction was accomplished through addition of 14 uL of 0.5 M tris(2-carboxyethyl)phosphine (TCEP) with incubation at rt for 2 h within a ThermoMixer (ThermoFisher) set at 1,200 RPM. This was followed by alkylation using 20 uL of 0.5 M iodoacetamide (IAA) and incubation for 30 min at rt in complete darkness at 1,200 RPM. The reaction was quenched through addition of 50 uL of dithiothreitol (DTT) before clarification of samples with 15 min centrifugation at 18,000 RCF at 10 deg C. The resulting supernatant was added to a 3 K MWCO Amicon Ultra 0.5 mL centrifugal filter (MilliporeSigma) and centrifuged at 14,000 RCF for 60 min at 10 deg C. Samples were analyzed using a Waters NanoACQUITY UPLC system with mobile phases consisting of 0.2% FA in H2O (Mobile Phase A) and 0.2% FA in ACN (Mobile Phase B). Both trapping-precolumn (150 um i.d., 5-cm length) and analytical column (100 um i.d., 50-cm length) were slurry-packed with C2 packing material (5 um and 3 um for trap/analytical respectively, 300 Angstrom, Separation Methods Technology). Samples were loaded into a 10 uL loop, corresponding to 400 ng of loaded material, and injected into the trapping column with an isocratic flow of 1% B at 5 uL/min over 15 min for desalting. Separation was performed with a 1% to 50% B gradient over 140 min at 300 nL/min. For MS/MS analysis of proteins, the NanoACQUITY system was coupled to a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with the FAIMS Pro interface. [doi:10.25345/C5G15TN11] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: FAIMS ; top-down proteomics ; pancreas ; diabetes ; insulin

Contact

Principal Investigators:
(in alphabetical order) 		Wei-Jun Qian, Pacific Northwest National Laboratory, United States
Submitting User: alchemistmatt
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 Quantification 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.