PXD007058

PXD007058 is an original dataset announced via ProteomeXchange.

Title	C4PR_LIV Evaluation of Parameters for Confident Phosphorylation Site Localization using an Orbitrap Fusion Tribrid Mass Spectrometer
Description	Confident identification of sites of protein phosphorylation by mass spectrometry (MS) is essential to advance understanding of phosphorylation-mediated signaling events. However, development of novel instrumentation requires that methods for MS data acquisition and its interrogation be evaluated and optimized for high throughput phosphoproteomics. Here, we compare and contrast eight MS acquisition methods on the novel tribrid Orbitrap Fusion MS platform, using both a synthetic phosphopeptide library and a complex phosphopeptide-enriched cell lysate. As well as evaluating multiple fragmentation regimes (HCD, EThcD and neutral loss triggered ET(ca/hc)D), and analyzers for MS/MS (orbitrap (OT) versus ion trap (IT)), we also compare two commonly used bioinformatics platforms, Andromeda with PTM-score, and MASCOT with ptmRS, for confident phosphopeptide identification and, crucially, phosphosite localization. Our findings demonstrate that optimal phosphosite identification is achieved using HCD fragmentation and high resolution orbitrap-based MS/MS analysis, employing MASCOT/ptmRS for data interrogation. Although EThcD is optimal for confident site localization for a given PSM, the increased duty cycle compared with HCD compromises the numbers of phosphosites identified. Finally, our data highlights that a charge-state dependent fragmentation regime, and a multiple algorithm search strategy, are likely to be of benefit for confident large-scale phosphosite localization.
HostingRepository	PRIDE
AnnounceDate	2017-08-10
AnnouncementXML	Submission_2017-08-10_00:30:48.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Samantha Ferries
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion ETD

Revision	Datetime	Status	ChangeLog Entry
0	2017-07-17 01:35:30	ID requested
1	2017-08-08 00:43:01	announced
⏵ 2	2017-08-10 00:30:49	announced	Updated project metadata.

Ferries S, Perkins S, Brownridge PJ, Campbell A, Eyers PA, Jones AR, Eyers CE, Evaluation of Parameters for Confident Phosphorylation Site Localization Using an Orbitrap Fusion Tribrid Mass Spectrometer. J Proteome Res, 16(9):3448-3459(2017) [pubmed]

curator keyword: Technical

submitter keyword: mass spectrometry, orbitrap fusion, phosphoproteomics, Mascot/ptmRS, Andromeda/PTMscore

Professor Claire E Eyers
contact affiliation	Institute of Integrative Biology University of Liverpool Crown Street, Liverpool L69 7ZB
contact email	ceyers@liverpool.ac.uk
lab head
Samantha Ferries
contact affiliation	University of Liverpool
contact email	s.ferries@liv.ac.uk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD007058
     1. Label: PRIDE project
     2. Name: C4PR_LIV Evaluation of Parameters for Confident Phosphorylation Site Localization using an Orbitrap Fusion Tribrid Mass Spectrometer