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  Benchmarking Multiple Fragmentation Methods on an Orbitrap Fusion for Top-down Phospho-Proteoform Characterization

Brunner, A. M., Lössl, P., Liu, F., Huguet, R., Mullen, C., Yamashita, M., et al. (2015). Benchmarking Multiple Fragmentation Methods on an Orbitrap Fusion for Top-down Phospho-Proteoform Characterization. ANALYTICAL CHEMISTRY, 87(8), 4152-4158. doi:10.1021/acs.analchem.5b00162.

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 Creators:
Brunner, Andrea M.1, Author
Lössl, Philip1, Author
Liu, Fan1, Author
Huguet, Romain1, Author
Mullen, Christopher1, Author
Yamashita, Masami2, Author              
Zabrouskov, Vlad1, Author
Makarov, Alexander1, Author
Altelaar, A. F. Maarten1, Author
Heck, Albert J. R.1, Author
Affiliations:
1external, ou_persistent22              
2Conti, Elena / Structural Cell Biology, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565144              

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Free keywords: ELECTRON-TRANSFER DISSOCIATION; TANDEM MASS-SPECTROMETRY; ULTRAVIOLET PHOTODISSOCIATION; CAPTURE DISSOCIATION; PEPTIDE; PROTEINS; AURORA; ETHCD; ACTIVATION; COVERAGE
 Abstract: 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. Data are available via ProteomeXchange with identifier PXD001845.

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Language(s): eng - English
 Dates: 2015
 Publication Status: Published in print
 Pages: 7
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

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Title: ANALYTICAL CHEMISTRY
Source Genre: Journal
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Publ. Info: 1155 16TH ST, NW, WASHINGTON, DC 20036 USA : AMER CHEMICAL SOC
Pages: - Volume / Issue: 87 (8) Sequence Number: - Start / End Page: 4152 - 4158 Identifier: ISSN: 0003-2700