PXD001845 is an
original dataset announced via ProteomeXchange.
Dataset Summary
Title | Benchmarking multiple fragmentation methods on an Orbitrap Fusion for top-down phospho-proteoform characterization |
Description | Top-down analysis of intact proteins by mass spectrometry provides an ideal platform for comprehensive proteoform characterization, in particular, for the identification and localization of post-translational modifications (PTM) co-occurring on a protein. One of the main bottlenecks in top-down proteomics is insufficient protein sequence coverage caused by incomplete protein fragmentation. Based on previous work on peptides, increasing sequence coverage and PTM localization by combining sequential ETD and HCD fragmentation in a single fragmentation event, we hypothesized that protein sequence coverage and phospho-proteoform characterization could be equally improved by this new dual fragmentation method termed EThcD, recently been made available on the Orbitrap Fusion. Here, we systematically benchmark the performance of several (hybrid) fragmentation methods for intact protein analysis on an Orbitrap Fusion, using as a model system a 17.5 kDa N-terminal fragment of the mitotic regulator Bora. During cell division Bora becomes multiply phosphorylated by a variety of cell cycle kinases, including Aurora A and Plk1, albeit at distinctive sites. Here, we monitor the phosphorylation of Bora by Aurora A and Plk1, analyzing the generated distinctive phospho-proteoforms by top-down fragmentation. We show that EThcD and ETciD on a Fusion are feasible and capable of providing richer fragmentation spectra compared to HCD or ETD alone, increasing protein sequence coverage, and thereby facilitating phosphosite localization and the determination of kinase specific phosphorylation sites in these phospho-proteoforms. |
HostingRepository | PRIDE |
AnnounceDate | 2015-07-16 |
AnnouncementXML | Submission_2015-07-16_06:42:17.xml |
DigitalObjectIdentifier | |
ReviewLevel | Peer-reviewed dataset |
DatasetOrigin | Original dataset |
RepositorySupport | Unsupported dataset by repository |
PrimarySubmitter | Andrea Mizzi Brunner |
SpeciesList | scientific name: Homo sapiens (Human); NCBI TaxID: 9606; |
ModificationList | phosphorylated residue |
Instrument | Orbitrap Fusion ETD |
Dataset History
Revision | Datetime | Status | ChangeLog Entry |
0 | 2015-02-25 06:03:42 | ID requested | |
⏵ 1 | 2015-07-16 06:42:19 | announced | |
Publication List
Brunner AM, L, รถ, ssl P, Liu F, Huguet R, Mullen C, Yamashita M, Zabrouskov V, Makarov A, Altelaar AF, Heck AJ, Benchmarking multiple fragmentation methods on an orbitrap fusion for top-down phospho-proteoform characterization. Anal Chem, 87(8):4152-8(2015) [pubmed] |
Keyword List
submitter keyword: Bora, Orbitrap Fusion, top down proteomics, EThcD, fragmentation, phosphorylation, proteoform |
Contact List
Albert J.R. Heck |
contact affiliation | Biomolecular Mass Spectrometry and Proteomics Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences Utrecht University Padualaan 8 3584 CH Utrecht The Netherlands |
contact email | A.J.R.Heck@uu.nl |
lab head | |
Andrea Mizzi Brunner |
contact affiliation | Utrecht University |
contact email | a.m.brunner@uu.nl |
dataset submitter | |
Full Dataset Link List
Dataset FTP location
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PRIDE project URI |
Repository Record List
[ + ]
[ - ]
- PRIDE
- PXD001845
- Label: PRIDE project
- Name: Benchmarking multiple fragmentation methods on an Orbitrap Fusion for top-down phospho-proteoform characterization