Abstract
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 library and a complex phosphopeptide-enriched cell lysate. In addition to 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 highlight 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.
Highlights
Protein phosphorylation is an essential, rapidly reversible, posttranslational modification (PTM) with critical roles in most biological processes
We have systematically evaluated eight mass spectrometry (MS) acquisition strategies on the Orbitrap Fusion mass spectrometer, a versatile tribrid MS platform, for their ability to confidently identify and, crucially, to pinpoint sites of modification on phosphopeptides
We have examined the relative efficiency of two of the most widely used phosphoproteomics data analysis platforms for optimal phosphosite identification: MASCOT integrated into Proteome Discover using ptmRS and Andromeda with PTM-score
Summary
Protein phosphorylation is an essential, rapidly reversible, posttranslational modification (PTM) with critical roles in most biological processes. Defining these dynamic phosphorylation events is key to understanding their functional significance and gaining insight into the complex biology that they regulate. Over the past decade have allowed for in-depth, arguably incomplete, analysis of phosphoproteomes in a wide variety of complex biological systems.[1−12] The continual development of more sophisticated ways of generating and analyzing MS data is undoubtedly aiding phosphopeptide identification. Reviewers and users of such data generally understand this importance, and publication guidelines typically require researchers to assess site localization confidence.[13]
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