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PXD000658

PXD000658 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleGlobal phosphoproteomic profiling reveals distinct signatures in B-cell non-Hodgkin lymphomas
DescriptionAmmonium hydroxide and pyrrolidine eluents were dried (SpeedVac) and reconstituted in 25 ul sample loading buffer (0.1% TFA/2% acetonitrile). Eluent from phosphotyrosine immunoprecipitation was dried and reconstituted in 35 ul of the loading buffer. An LTQ Orbitrap XL (ThermoFisher) in-line with a Paradigm MS2 HPLC (Michrom bioresources) was employed for acquiring high-resolution MS and MS/MS data. Ten microliters of the phospho-enriched peptides were loaded onto a sample trap (Captrap, Bruker-Michrom) in-line with a nano-capillary column (Picofrit, 75 um i.d.x 15 um tip, New Objective) packed in-house with 10 cm of MAGIC AQ C18 reverse phase material (Michrom Bioresource). Two different gradient programs, one each for MOAC and phosphotyrosine immunoprecipitation samples, were used for peptide elution. For MOAC samples, a gradient of 5-40% buffer B (95% acetonitrile/1% acetic acid) in 135 min and 5 min wash with 100% buffer B followed by 30 min of re-equilibration with buffer A (2% acetonitrile/1% acetic acid) was used. For phosphotyrosine immunoprecipitation samples, which were a much less complex mixture of peptides, 5-40% gradient with buffer B was achieved in 75 min followed by 5 min wash with buffer B and 30 min re-equilibration. Flow rate was ~0.3 ul/min. Peptides were directly introduced into the mass spectrometer using a nano-spray source. Orbitrap was set to collect 1 MS scan between 400-2000 m/z (resolution of 30,000 @ 400 m/z) in orbitrap followed by data dependent CID spectra on top 9 ions in LTQ (normalized collision energy ~35%). Dynamic exclusion was set to 2 MS/MS acquisitions followed by exclusion of the same precursor ion for 2 min. Maximum ion injection times were set to 300 ms for MS and 100 ms for MS/MS. Automatic Gain Control (AGC) was set to 1xe6 for MS and 5000 for MS/MS. Charge state screening was enabled to discard +1 and unassigned charge states. Technical duplicate data for each of the MOAC elutions (ammonium hydroxide and pyrrolidine) and triplicate data for the phosphotyrosine immunoprecipitation samples were acquired. RAW files were converted to mzXML using msconvert and searched against the Swissprot Human protein database (2013-Jan release) appended with common proteomics contaminants and reverse sequences as decoys. Searches were performed with X!Tandem (version 2010.10.01.1) using the k-score plugin. For all searches the following search parameters were used: Parent monoisotopic mass error of 50 ppm; fragment ion error of 0.8 Daltons; allowing for up to 2 missed tryptic cleavages. Variable modifications were oxidation of Methionine (+15.9949@M), carbamidomethylation of Cysteine (+57.0214@C), and phosphorylation of Serine, Threonine, and Tyrosine (+79.9663@[STY]). The search results were then post-processed using PeptideProphet and ProteinProphet.
HostingRepositoryPRIDE
AnnounceDate2014-07-25
AnnouncementXMLSubmission_2014-08-08_06:05:27.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterDamian Fermin
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListmonohydroxylated residue; phosphorylated residue; iodoacetamide derivatized residue
InstrumentLTQ Orbitrap XL
Dataset History
RevisionDatetimeStatusChangeLog Entry
02013-12-19 02:05:29ID requested
12014-01-09 08:27:54announced
22014-01-10 01:32:23announcedRunning publication again through PRIDE
32014-01-10 02:00:20announcedRunning publication again through PRIDE
42014-07-25 01:51:30announcedUpdated project metadata.
52014-08-08 06:05:28announcedUpdated project metadata.
Publication List
Rolland D, Basrur V, Conlon K, Wolfe T, Fermin D, Nesvizhskii AI, Lim MS, Elenitoba-Johnson KS, Global phosphoproteomic profiling reveals distinct signatures in B-cell non-Hodgkin lymphomas. Am J Pathol, 184(5):1331-42(2014) [pubmed]
Keyword List
submitter keyword: MS/MS,B-cell lymphoma,phosphoproteome
Contact List
Damian Fermin
contact affiliationPathology Department, University of Michigan
contact emaildfermin@umich.edu
dataset submitter
Full Dataset Link List
Dataset FTP location
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PRIDE project URI
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