PXD009252-2

PXD009252 is an original dataset announced via ProteomeXchange.

Title	A mass spectrometry-based predictive strategy reveals ADAP1 is phosphorylated at Tyrosine 364.
Description	Abstract Rationale The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry-based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition. Methods ADAP1 was immunoprecipitated from extracts of HEK293 cells stably-transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC-MS in MS1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data mining algorithm. Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC-MS2. Acquired MS2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Results Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a Tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form. Conclusions A novel phosphorylation site was identified at Tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that Tyrosine 364 is phosphorylated by a PKC-dependent mechanism.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_04:43:58.833.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD009252
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Rick Reisdorph
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	Phospho; Oxidation
Instrument	6520A Quadrupole Time-of-Flight LC/MS

Revision	Datetime	Status	ChangeLog Entry
0	2018-03-19 05:24:39	ID requested
1	2018-04-26 06:10:58	announced
⏵ 2	2024-10-22 04:44:07	announced	2024-10-22: Updated project metadata.

Reisdorph R, Littrell-Miller B, Powell R, Reisdorph N, A mass spectrometry based predictive strategy reveals ADAP1 is phosphorylated at tyrosine 364. Rapid Commun Mass Spectrom, 32(15):1173-1180(2018) [pubmed]

10.6019/PXD009252;

10.1002/rcm.8140;

curator keyword: Biological

submitter keyword: phosphorylation,ADAP1

Richard M. Reisdorph
contact affiliation	University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences
contact email	richard.reisdorph@ucdenver.edu
lab head
Rick Reisdorph
contact affiliation	University of Colorado, Skaggs School of Pharmacy and Pharmaceutical Sciences
contact email	richard.reisdorph@ucdenver.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD009252
     1. Label: PRIDE project
     2. Name: A mass spectrometry-based predictive strategy reveals ADAP1 is phosphorylated at Tyrosine 364.