Dataset Information

C4PR_LIV Evaluation of Parameters for Confident Phosphorylation Site Localization using an Orbitrap Fusion Tribrid Mass Spectrometer

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Bone Osteosarcoma

SUBMITTER: Samantha Ferries

LAB HEAD: Professor Claire E Eyers

PROVIDER: PXD007058 | Pride | 2019-09-27

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	SF_200217_U2OS_TiO2_EThcD_IT_rep1.msf	Msf
	SF_200217_U2OS_TiO2_EThcD_IT_rep1.raw	Raw
	SF_200217_U2OS_TiO2_EThcD_IT_rep2.msf	Msf
	SF_200217_U2OS_TiO2_EThcD_IT_rep2.raw	Raw
	SF_200217_U2OS_TiO2_EThcD_OT_rep1.msf	Msf

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Publications

Evaluation of Parameters for Confident Phosphorylation Site Localization Using an Orbitrap Fusion Tribrid Mass Spectrometer.

Ferries Samantha S Perkins Simon S Brownridge Philip J PJ Campbell Amy A Eyers Patrick A PA Jones Andrew R AR Eyers Claire E CE

Journal of proteome research 20170811 9

Confident identification of sites of protein phosphorylation by mass spectrometry (MS) is essential to advance understanding of phosphorylation-mediated signaling events. However, the 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 ...[more]

PMID: 28741359

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