MassIVE MSV000097121

Complete Public PXD060835

Analysis of a cancer-associated mutation in the budding yeast Nuf2 kinetochore protein

Subscribe Comment Convert Spectra Reanalyze Spectra Compare Results Add Reanalysis

Description

Saccharomyces cerevisiae kinetochores were isolated from a NUF2-3V5 background via Dsn1-His-Flag purifications. (AAL020924_02154_SBY23028) and (AAL020924_021524_SBY23066) were grown at 30 C and harvested once cell cultures reached OD600nm = ~3. Cells were resuspended in Buffer H (25mM HEPES pH8.0, 2mM MgCl2 , 0.1 mM EDTA pH 8.0, 0.5 mM EGTA-KOH pH8.0, 15% Glycerol, 0.1% NP-40, and 150 mM KCl) containing phosphatase inhibitors (1 mM sodium-pyrophosphate, 2 mM sodium-beta-glycerophosphate, 0.1 mM sodium orthovanadate, and 5 mM NaF), 0.1 mM microcystin-LR, 0.2 mM PMSF, and protease inhibitors (10 mg/mL for each of the following leupeptin, pepstatin A, and chymostatin). In which cells were spun down and flash frozen in liquid nitrogen. Pellets were lysed via Freezer Mill (SPEX SamplePrep) and treated with 50 units/ml of benzonase (EMD Milipore Corp) for 30 minutes on ice. Lysate proteins were extracted after undergoing ultracentrifugation at 24,000 RPM for 90 minutes at 4 °C. The Pierce BCA Protein Assay Kit (Thermo Scientific) was used to measure the protein concentration within each extract which were subsequently normalized. Extracts were incubated with a-M3DK (GenScript) magnetic Protein G Dynabeads (Invitrogen) for 3 hours at 4 °C with rotation. Beads were washed three times with BH0.15 containing phosphatase inhibitors, protease inhibitors, 0.1 mM microcystin-LR, and 2 mM DTT. Then two times with BH0.15 containing protease inhibitors and LPC. Then rinsed two times with pre-elution rinse buffer (50 mM Tris pH 8.3, 75 mM KCl, and 1 mM EGTA). Purified kinetochores were eluted from beads into 70 ml of 0.2% RapiGest (Waters Corporation) in 50 mM ammonium bicarbonate. This was achieved via gentle agitation on a Vortex-Genie 2 (Scientific Industries) set to the lowest setting for 30 minutes at room temperature. 60 ml of each elution sample was sent for mass spectrometry processing. [doi:10.25345/C5NV99P5J] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Saccharomyces cerevisiae ; kinetochores ; phosphorylation ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		Susan Biggins, Fred Hutchinson Cancer Center, USA
Submitting User: FHproteomics
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.