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Enrichment and Separation of Phosphorylated Peptides on Titanium Dioxide Surfaces: Applied and Fundamental Studies
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
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 [en]
Posttranslational modification, Phosphorylation, Mass spectrometry, MALDI, Adsorption, QCM-D
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-204723ISBN: 978-91-554-8717-1 (print)OAI: oai:DiVA.org:uu-204723DiVA: diva2:639881
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
List of papers
1. Optimized Protocol for On-Target Phosphopeptide Enrichment Prior to Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry Using Mesoporous Titanium Dioxide
Open this publication in new window or tab >>Optimized Protocol for On-Target Phosphopeptide Enrichment Prior to Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry Using Mesoporous Titanium Dioxide
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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.

National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:uu:diva-125767 (URN)10.1021/ac100589j (DOI)000278062800040 ()20443553 (PubMedID)
Available from: 2010-05-31 Created: 2010-05-28 Last updated: 2017-12-12Bibliographically approved
2. 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
Open this publication in new window or tab >>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
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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.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-148662 (URN)10.1021/ac1027879 (DOI)000286689600021 ()21210638 (PubMedID)
Available from: 2011-03-09 Created: 2011-03-09 Last updated: 2017-12-11Bibliographically approved
3. Physicochemical Characterization of Phosphopeptide/Titanium Dioxide Interactions Employing the Quartz Crystal Microbalance Technique
Open this publication in new window or tab >>Physicochemical Characterization of Phosphopeptide/Titanium Dioxide Interactions Employing the Quartz Crystal Microbalance Technique
2013 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 7, 2019-2025 p.Article in journal (Refereed) Published
Abstract [en]

The rapidly growing field of phosphoproteomics has led to a strong demand for procedures enabling fast and reliable isolation and enrichment of phosphorylated proteins and peptides. During the past decade, several novel phosphopeptide enrichment methods based on the affinity of phosphoryl groups for titanium dioxide (TiO2) have been developed and tested. The ultimate goal of obtaining comprehensive phosphoproteomes has, however, been found difficult to achieve and the obtained results often vary, dependent on the enrichment method and protocol used. In the present study, the physical chemistry of the phosphopeptide binding to TiO2 is investigated by means of measurements using a quartz crystal microbalance with dissipation monitoring (QCM-D). Special emphasis is put on the effect of the degree of phosphorylation of the phosphopeptide, the impact of the primary amino acid structure, and the role of electrostatic interactions. The results show that, in general, adsorption of phosphopeptides follows the Langmuir model and that the affinity for the TiO2 surface increases in a nonlinear fashion with increasing degree of phosphorylation. An exception was detected, however, where positive cooperativity between the peptides existed and the Langmuir model no longer applied. The source behind the cooperativity could be traced back to the primary amino acid structure and, more specifically, the presence of positively charged amino acids in positions that enable electrostatic interaction with phosphoryl groups on neighboring peptides. Regardless of the net peptide charge, the TiO2–phosphopeptide interaction was for all phosphopeptides investigated found to be mainly of electrostatic origin. This study highlights and explains some of the most common problems with the TiO2-based enrichment methods used today.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2013
National Category
Physical Chemistry
Research subject
Chemistry with specialization in Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-195262 (URN)10.1021/jp310161m (DOI)000315432200004 ()
Available from: 2013-02-22 Created: 2013-02-22 Last updated: 2017-12-06Bibliographically approved
4. On-target titanium dioxide-based enrichment for characterization of phosphorylations in the Adenovirus pIIIa protein
Open this publication in new window or tab >>On-target titanium dioxide-based enrichment for characterization of phosphorylations in the Adenovirus pIIIa protein
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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.

Keyword
Phosphopeptide enrichment, MALDI-MS, Separation, Capsid protein precursor pIIIa, TiO2
National Category
Analytical Chemistry Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-204683 (URN)10.1016/j.chroma.2013.08.096 (DOI)000327229600012 ()
Available from: 2013-08-09 Created: 2013-08-08 Last updated: 2017-12-06Bibliographically approved

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