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 (ncbitaxon:9606)

SUBMITTER: Professor Claire E Eyers  

PROVIDER: MSV000085203 | MassIVE | Tue Mar 31 06:59:00 BST 2020

SECONDARY ACCESSION(S): PXD007058

REPOSITORIES: MassIVE