PXD060835

PXD060835 is an original dataset announced via ProteomeXchange.

Title	Analysis of a cancer-associated mutation in the budding yeast Nuf2 kinetochore protein
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.
HostingRepository	MassIVE
AnnounceDate	2025-03-10
AnnouncementXML	Submission_2025-03-10_12:58:30.263.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Fred Hutch Proteomics Core
SpeciesList	scientific name: Saccharomyces cerevisiae W303; NCBI TaxID: 580240;
ModificationList	Phospho
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2025-02-14 11:20:27	ID requested
⏵ 1	2025-03-10 12:58:30	announced

no publication

submitter keyword: Saccharomyces cerevisiae, kinetochores, phosphorylation, DatasetType:Proteomics

Susan Biggins
contact affiliation	Fred Hutchinson Cancer Center
contact email	sbiggins@fredhutch.org
lab head
Fred Hutch Proteomics Core
contact affiliation	Fred Hutchinson Cancer Research Center
contact email	pgafken@fredhutch.org
dataset submitter

MassIVE dataset URI

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