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On-target titanium dioxide-based enrichment for characterization of phosphorylations in the Adenovirus pIIIa protein
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
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2013 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1317, no SI, 105-109 p.Article in journal (Refereed) Published
Abstract [en]

A recently developed titanium dioxide (TiO2) based on-target method for phosphopeptide enrichment and matrix assisted laser desorption-ionization mass spectrometry (MALDI MS) analysis was used to investigate phosphorylations in the Adenovirus type 2 structural protein pIIIa. Lysates of purified virus particles were separated on 1-D SDS-PAGE and the band for the pIIIa protein was excised for tryptic digestion into peptides that were enriched with the on-target method. The enrichment provided by the method clearly improved the detectability of phosphorylated peptides and the results show for the first time evidence for multi-phosphorylated peptides in pIIIa. Moreover, three novel phosphorylations were identified in the protein sequence, even though the precise positions could not be determined. These results illustrate the potential of the method for the characterization of novel phosphoproteomes in biological samples of medical relevance.

Place, publisher, year, edition, pages
2013. Vol. 1317, no SI, 105-109 p.
Keyword [en]
Phosphopeptide enrichment, MALDI-MS, Separation, Capsid protein precursor pIIIa, TiO2
National Category
Analytical Chemistry Physical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-204683DOI: 10.1016/j.chroma.2013.08.096ISI: 000327229600012OAI: oai:DiVA.org:uu-204683DiVA: diva2:639806
Available from: 2013-08-09 Created: 2013-08-08 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Enrichment and Separation of Phosphorylated Peptides on Titanium Dioxide Surfaces: Applied and Fundamental Studies
Open this publication in new window or tab >>Enrichment and Separation of Phosphorylated Peptides on Titanium Dioxide Surfaces: Applied and Fundamental Studies
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein phosphorylation is a very common posttranslational modification (PTM), which lately has been found to hold the keyrole in the development of many severe diseases, including cancer. Thereby, phosphoprotein analysis tools, generally based on specific enrichment of the phosphoryl group, have been a hot topic during the last decade.

In this thesis, two new TiO2-based on-target enrichment methods are developed and presented together with enlightening fundamental results.

Evaluation of the developed methods was performed by the analysis of: custom peptides, β-casein, drinking milk, and the viral protein pIIIa. The results show that: i) by optimizing the enrichment protocol (first method), new phosphorylated peptides can be found and ii) by the addition of a separation step after the enrichment (second method), more multi-phosphorylated peptides, which usually are hard to find, could be detected. The fundamental part, on the other hand, shows that the phosphopeptide adsorption is caused by electrostatic interactions, in general follows the Langmuir model, and the affinity increases with the phosphorylation degree. Here, however, the complexity of the system was also discovered, as the adsorption mechanism was found to be affected by the amino acid sequence of the phosphopeptide.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1059
Keyword
Posttranslational modification, Phosphorylation, Mass spectrometry, MALDI, Adsorption, QCM-D
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-204723 (URN)978-91-554-8717-1 (ISBN)
Public defence
2013-09-27, The Svedbergssalen, BMC, Husargatan 3, Uppsala, 10:00 (English)
Opponent
Supervisors
Available from: 2013-09-06 Created: 2013-08-09 Last updated: 2014-01-07

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Eriksson, AnnaBergquist, JonasEdwards, KatarinaBergström Lind, SaraAgmo Hernández, Víctor

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