PXD000360

PXD000360 is an original dataset announced via ProteomeXchange.

Title	3xMBT pull-down from wild-type and Rkm1 knock-out yeast
Description	We have presented a protocol that utilizes the triple malignant brain tumor domains of L3MBTL1 (3xMBT) to enrich proteins modified with mono- and di-methylated lysine from cell lysate. Cells in culture are grown with amino acids containing light or heavy stable isotopic labels. Methylated proteins are enriched by incubating cell lysates with 3xMBT, or with the binding-null D355N mutant as a negative control. Quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) is then used to identify proteins that are specifically enriched by 3xMBT pull-down. Addition of a third isotopic label allows comparison of protein methylation between biological conditions. As an example yeast cells were prepared in SILAC media under three conditions. In two independent experiments either light or heavy cells are Rkm1 knock-out, while the other two are wild-type. 3xMBT was used to enrich methylated proteins from the light/heavy cells, with 3xMBT_D355N as a negative control using the medium cell lysate. Proteins were separated into three fractions by SDS-PAGE and analyzed by LC-MS/MS using an Orbitrap Velos. Data was analyzed with MaxQuant version 1.3.0.5 using default parameters except that mono- and di-methylation of lysine were included as variable modifications, the modification-specific false-discovery was set to 10%, and minimum peptide count for quantitative data set to 1. The amount of ribosomal protein Rpl23, a known substrate of Rkm1, is greatly decreased in 3xMBT pull-down from Rkm1 knock-out cells relative to wild-type. The level of heat shock protein SSA4 is also greatly reduced, suggesting that it may also be methylated by Rkm1.
HostingRepository	PRIDE
AnnounceDate	2014-07-25
AnnouncementXML	Submission_2014-07-25_01:25:15.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Scott Carlson
SpeciesList	scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: 4932;
ModificationList	N6: N6-dimethyl-L-lysine; monomethylated L-lysine
Instrument	LTQ Orbitrap Elite

Revision	Datetime	Status	ChangeLog Entry
0	2013-07-24 02:11:34	ID requested
1	2013-11-11 03:46:32	announced
⏵ 2	2014-07-25 01:25:16	announced	Updated project metadata.

Dataset with its publication pending

submitter keyword: Methylation, lysine, L3MBTL1, Rkm1

Scott Carlson
contact affiliation	Biology
contact email	scottmc@stanford.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD000360
     1. Label: PRIDE project
     2. Name: 3xMBT pull-down from wild-type and Rkm1 knock-out yeast