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, 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.

Extensive non-canonical phosphorylation in human cells revealed using strong-anion exchange-mediated phosphoproteomics

“…Consequently, this triplet neutral loss pattern could theoretically be exploited to improve identification of pHis-containing peptides from LC-MS/MS data. In agreement with these findings, we observed characteristic triplet neutral losses in the tandem mass spectra of all five pHis myoglobin peptides following both ion-trap CID and higher energy collision dissociation (HCD), which represents the optimal fragmentation regime for phosphopeptide identification using the Orbitrap platform 41 . To investigate the utility of triplet neutral loss as a potential signature for accurate pHis site localisation and high-throughput pHis characterisation in complex samples, we assessed precursor neutral loss for phosphopeptides with confident phosphosite localisation (1% FLR) derived from a human HeLa cell lysate known to contain pSer, pThr, pTyr and pHis, using either CID or HCD-based fragmentation ( Fig.…”

Strong anion exchange‐mediated phosphoproteomics reveals extensive human non‐canonical phosphorylation

“…Immunoblotting of cell extracts confirmed efficient knockdown of PHPT1 in HeLa cells (Fig B), from which UPAX was subsequently performed. Sixteen SAX fractions were collected and analysed by LC‐MS/MS using HCD nlEThcD, where precursor ions exhibiting neutral loss of 98 amu following HCD were subjected to EThcD fragmentation (Frese et al , ; Ferries et al , ). A representative UV trace for the SAX separation (Fig C) and associated base peak chromatograms for three of the 16 pooled SAX fractions are shown (Fig D–F).…”

“…In contrast, there was a marginal decrease in the number of phosphopeptides identified irrespective of the residue type, suggesting that the functions of PHPT1 as a pHis (or potentially pLys) phosphatase are likely to be redundant. For comparison, we applied the same LC-MS/MS acquisition strategy and data interrogation pipeline to analyse a TiO 2 -enriched phosphopeptide set prepared using a more "typical" phosphoproteome workflow (Ferries et al, 2017) from HeLa cells ( Fig 3C and Appendix Table S6). While~41% of the phosphorylation sites identified at ptmRS 0.75 were assigned to non-canonical residues in the UPAX dataset, this decreased to 4.4% in the phosphopeptides enriched using TiO 2 , demonstrating the necessity to maintain nearneutral pH during sample preparation for the identification of noncanonical phosphosites.…”

PTMProphet: Fast and Accurate Mass Modification Localization for the Trans-Proteomic Pipeline

“…To demonstrate the performance of PTMProphet, we ran the tool against three example datasets, i) a small reference dataset of synthetic peptides previously published and used to evaluate confident site localization with an Thermo Fisher Scientific Orbitrap Fusion Tribrid mass spectrometer (Dataset #1, Ferries et al 2017) 17 , ii) a large in-house generated and unpublished dataset of synthetic phosphopeptides with known phosphorylated sites (Dataset #2), and iii) a phosphoenriched cell lysate dataset known to contain many mass modifications (Dataset #3, Söderholm et al) 43 .…”

“…Furthermore, based on this analysis and as displayed in Figure 2A Next, we compared the performance of PTMProphet to the originally published results of this dataset. While we can't compare PTMProphet in detail to all available site localization tools with their different strengths and weaknesses, we compare it here to ptmRS 17 and PTMiner, a pair of representative recently developed and well-performing PTM localization tools. We wanted to compare PTMProphet to the best performing tools and thus used the best performing dataset from the best performing method, HCD Orbitrap, and analysis tool, ptmRS, from the Ferries et al 2017 publication.…”