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Optimized Protocol for On-Target Phosphopeptide Enrichment Prior to Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry Using Mesoporous Titanium Dioxide
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
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2010 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 82, no 11, 4577-4583 p.Article in journal (Refereed) Published
Abstract [en]

A novel on-target phosphopeptide enrichment method is presented that allows specific enrichment and direct analysis by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) of phosphorylated peptides. Spots consisting of a thin film of anatase titanium dioxide are sintered onto a conductive glass surface. Enrichment and analysis can be performed on the modified target with minimal sample handling. The protocol leads to an enrichment efficiency that is superior to what has been reported before for similar methods. The method was tested using beta-casein as a model phosphorylated protein as well as with a custom peptide mixed with its phosphorylated form. A very low detection limit, a significantly improved phosphoprofiling capability, and a simple experimental approach provide a powerful tool for the enrichment, detection, and analysis of phosphopeptides.

Place, publisher, year, edition, pages
2010. Vol. 82, no 11, 4577-4583 p.
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-125767DOI: 10.1021/ac100589jISI: 000278062800040PubMedID: 20443553OAI: oai:DiVA.org:uu-125767DiVA: diva2:321000
Available from: 2010-05-31 Created: 2010-05-28 Last updated: 2014-01-07Bibliographically 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, KatarinaHagfeldt, AndersMalmström, DavidAgmo Hernández, Víctor

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