期刊
JOURNAL OF PROTEOME RESEARCH
卷 16, 期 9, 页码 3448-3459出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.7b00337
关键词
mass spectrometry; phosphoproteomics; phosphosite localization; phosphorylation site; Orbitrap Fusion; ptmRS; PTM-score; MASCOT; Andromeda; EThcD
资金
- NorthWest Cancer Research [CR1088]
- Biotechnology and Biological Sciences Research Council (BBSRC) [BB/L009501/1, BB/M025705/1]
- BBSRC DTP Ph.D. studentship award
- Biotechnology and Biological Sciences Research Council [BB/M012557/1, BB/L005239/1, BB/L009501/1, BB/M023818/1, BB/M025705/1, 1510759] Funding Source: researchfish
- BBSRC [BB/M025705/1, BB/M023818/1, BB/L005239/1, BB/M012557/1, BB/L009501/1] Funding Source: UKRI
- MRC [G120/1030] Funding Source: UKRI
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 phosphorites 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.
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