PXD015705

PXD015705 is an original dataset announced via ProteomeXchange.

Title	Mudit analyses of the proteins substrates candidate of NRBP1 using TR-TUBE system
Description	293 stable cell line expressing NRBP1 or NRBP1 mutant were transfected with HA-TR-TUBE using TR-TUBE system to protect the degradation of the ubiquitin chains of the potential ligase substrate. The complexes were pulled down using anti-HA antibody and subsequently digested with modified Trypsin. Anti-diGly Antibody was used for isolation of ubiquitinated peptides which recognizes the Gly-Gly dipeptide bond attached to lysine. Enriched peptide pellets were dissolved in 100 micro liter of buffer A, 5 percent acetonitrile, 0.1 percent formic acid. Samples were pressure loaded on split-triple-phase fused-silica micro-capillary columns and analyzed by nanoflow liquid chromatography using an ultimate 3000 RSLC nano system coupled to a Q Exactive plus mass spectrometer equipped with a nanospray Flex Ion source, Thermo Fisher Scientific and analyzed using a 10-step MudPIT analysis. The MS-MS datasets were searched using ProLuCID. The samples were searched against a database of 146186 sequences, consisting of 72956 H. sapiens non-redundant proteins NCBI, 2015-03-25, 193 usual contaminants -such as human keratins, IgGs, and proteolytic enzymes, and the NRBP1-muant protein sequences. To estimate false discovery rates, each protein sequence was randomized leading to a total search space of 73091 sequences. The precursor and fragment mass tolerances were set to 10 ppm and 100 ppm, respectively. Diglycine modification of Lys side chains, Ser-Thr-Tyr phosphorylation, pyroglutamate formation, acetylation, methionine oxidation and cysteine methylthio were set as variable modifications. Peptide-spectrum matches were sorted, selected and compared using DTASelect together with in-house software swallow and sandmartin. Proteins had to be detected by at least 2 spectra and average FDRs at the protein and spectral levels were set to less than 4 percent. To estimate relative protein levels, Normalized Spectral Abundance Factors,dNSAFs, were calculated for each detected protein. NSAF7 was used to extract total and modified label-free features for each amino acid within and calculate modification levels based on local spectral counts and generate the modification results.
HostingRepository	MassIVE
AnnounceDate	2021-01-28
AnnouncementXML	Submission_2021-01-28_18:16:17.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Anita Saraf
SpeciesList	scientific name: Homo sapiens; common name: human; NCBI TaxID: 9606;
ModificationList	ubiquitination signature dipeptidyl lysine; L-methionine sulfoxide; L-cysteine methyl disulfide; O-phospho-L-serine; O-phospho-L-threonine; O4'-phospho-L-tyrosine; Gln->pyro-Glu
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2019-10-04 10:58:58	ID requested
⏵ 1	2021-01-28 18:16:17	announced

no publication

submitter keyword: E3 ubiquitin ligase, ubiquitination, protein degradation

Anita Saraf
contact affiliation	The Stowers Institute for Medical Research
contact email	ans@stowers.org
lab head
Anita Saraf
contact affiliation	Stowers Institute for Medical Research
contact email	ans@stowers.org
dataset submitter

MassIVE dataset URI

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