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Mesoporous TiO2-Based Experimental Layout for On-Target Enrichment and Separation of Multi- and Monophosphorylated Peptides Prior to Analysis with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
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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2011 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 83, no 3, 761-766 p.Article in journal (Refereed) Published
Abstract [en]

A simple method for on-target enrichment and subsequent separation and analysis of phosphorylated peptides is presented. The tryptic digest of a phosphorylated protein, in this case beta-casein, is loaded onto a spot on a thin stripe made of mesoporous TiO2 sintered onto a conductive glass surface. After washing with a salicylic buffer in order to remove the nonphosphorylated peptides, the stripe is placed in an elution chamber containing a phosphate solution. In a way analogous to thin layer chromatography (TLC), the phosphate solution acts as an eluent, clearly separating multi- and monophosphorylated peptides. By performing matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) along the stripe, the detection of all phosphorylated peptides present in the digest is facilitated, as they are isolated from each other. The method was also tested on commercial drinking milk, achieving successful separation between multi- and monophosphorylated peptides, as well as a detection limit in the femtomole range. As the enrichment, separation, and analysis take place in the same substrate, sample handling and risk of contamination and sample loss is minimized. The results obtained suggest that the method, once optimized, may successfully provide a complete phosphoproteome.

Place, publisher, year, edition, pages
2011. Vol. 83, no 3, 761-766 p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-148662DOI: 10.1021/ac1027879ISI: 000286689600021PubMedID: 21210638OAI: oai:DiVA.org:uu-148662DiVA: diva2:402648
Available from: 2011-03-09 Created: 2011-03-09 Last updated: 2014-01-07Bibliographically approved
In thesis
1. Interfacing Complementary Separation Techniques with Mass Spectrometry Utilizing Electrophoresis, Nanoparticles, and Functionalized Magnetic Beads
Open this publication in new window or tab >>Interfacing Complementary Separation Techniques with Mass Spectrometry Utilizing Electrophoresis, Nanoparticles, and Functionalized Magnetic Beads
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Capillary electrophoresis (CE) has during the last two decades become more robust and been able to separate neutral analytes without compromising the downstream detection. An interesting aspect in CE compared to more commonly used high-performance liquid chromatography is the orthogonal separation mechanism provided by CE. Compounds are separated based on charge and size with extremely high separation efficiencies. However, since mass spectrometry (MS) has become one of the most important analytical detectors and play a key role for pharmaceutical- and in clinical applications it is of major importance that the two techniques successfully can be combined without any compromises. Improvements in existing ion sources must be made in order to fully take advantage of the potential in capillary electrophoresis and mass spectrometry. One way is to miniaturize the ion source (paper I) in order to make it more compatible with the smaller liquid volumes and lower flow rates in CE. Despite these improvements challenges such as low sample concentrations, non-separated peaks, unspecific losses, and poor ionization still remain, and are addressed in this doctoral thesis.

Separation of neutral analytes has previously been achieved with packed columns but with several disadvantages. Therefore, MS-compatible pseudostationary phases in the form of nanoparticles (paper II) are an interesting alternative with its minimized risk of clogging, reduced memory effects and better separation efficiencies. Particles or beads have also shown to be of importance when reducing the dynamic range in complex samples. By creating functionalized magnetic beads (paper III), complex samples such as human plasma can be fractionated in the manner that low molecular weight proteins are selectively enriched. Despite fractionation and enrichment of analytes of interest (paper IV) the ionization suppression could lead to biased sensitivity, increased baseline, retention variations and chromatographic distortion. Therefore the separation, as well as the ionization, is of major importance. For instance, in order to separate and detect monoclonal antibodies, which are an upcoming class of biotherapeutic drugs, the choice of capillary temperature and sheath liquid composition must be considered due to its major influence on charge state, peak intensity and memory effects (paper V).

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 63 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 993
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-183666 (URN)978-91-554-8529-0 (ISBN)
Public defence
2012-12-14, B42, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2012-11-22 Created: 2012-10-31 Last updated: 2013-02-11Bibliographically approved
2. 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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