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Monomer surface modifications for rapid peptide analysis by capillary electrphoresis and capillary electrochromatography coupled to electrospray ionization mass spectrometry
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
2004 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 6, 809-816 p.Article in journal (Refereed) Published
Abstract [en]

In this study, positively charged alkylaminosilyl monomers were used to modify the inner surface of fused silica capillaries, which subsequently were employed in capillary electrophoresis (CE) and capillary electrochromatography (CEC). The obtained surfaces yield a reversed electroosmotic flow (EOF) and have varying carbon chain lengths, that interact with the analytes and give chromatographic retention. The coating procedure is very simple and fast. The performance of the modified capillaries was evaluated regarding pH influence on EOF and chromatographic interactions. The experiments were conducted with UV and mass spectrometry (MS) and applied to the separation of various neuropeptides. The derivatized surfaces showed a linear (R2 ∼0.99) pH dependence with isoelectric points (pI) at 8.6–8.8. Rapid separations of peptide standards and a protein digest with efficiencies as high as 5×105 plates/m were performed.

Place, publisher, year, edition, pages
2004. Vol. 25, no 6, 809-816 p.
Keyword [en]
Capillary electrochromatography, Capillary electrophoresis, Mass spectrometry, Peptide, Surface modification
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-94824DOI: 10.1002/elps.200305719OAI: oai:DiVA.org:uu-94824DiVA: diva2:168815
Available from: 2006-09-15 Created: 2006-09-15 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Column Development in Capillary Electrophoresis and Electrochromatography for Bioanalytical Applications
Open this publication in new window or tab >>Column Development in Capillary Electrophoresis and Electrochromatography for Bioanalytical Applications
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Analysis of biological samples can be a difficult task. This thesis covers a broad aspect of the analytical areas of capillary electrophoresis (CE) and capillary electrochromatography (CEC) in combination with mass spectrometry (MS) that are of great importance for achieving fast, accurate and sensitive bioanalyses. A significantly time reduced and automated system for sample cleanup was developed to greatly simplify the pretreatment process of biological samples with a complex matrix. Desalting and preconcentration of species in urine was conducted and the limit of detection for the antidepressant escitalopram was lowered 10 times. This extraction devise was also successfully incorporated in a chip based platform for the possibility to be a part of multidimensional separation systems. The reduced risk of sample loss leads to improved detection limits, which are usually one the most challenging parts when working with bioanalyses.

In the area of separation, a monomer surface with tailored hydrophobicity was developed to achieve rapid, high efficient separations of complex mixtures. Within five minutes a tryptic digest of a protein could be separated and then identified by a Mascot search.

The applications addressed have been focused on medical conditions which are of highest interest for both physicians and patients. A high throughput analysis of the kynurenine metabolites with CE-MS offers a new method to rapidly examine samples from patients with neurological disorders.

A screening study of possible biomarkers for the two different types of appendicitis, gangraenous and phlegmonous was conducted. Indicative patterns were found for both pre and post surgery of the two types of inflammation as well as between them. The divergences were traced back to the MS peaks obtained in the CE- and CEC-MS setups as possible biomarkers for the two forms of appendicitis. A preliminary study of polycystic ovary syndrome also offered some valuable results for future biomarker identification.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 216
Keyword
Analytical chemistry, Capillary electrophoresis (CE), Capillary electrochromatography (CEC), Mass spectrometry (MS), Bioanalysis, Sample pretreatment, Sol-gel, Analytisk kemi
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-7124 (URN)91-554-6644-3 (ISBN)
Public defence
2006-10-06, B22, BMC, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2006-09-15 Created: 2006-09-15 Last updated: 2012-02-08Bibliographically approved
2. Electrifying the Molecules of Life: Peptide and Protein Analysis by Capillary Electrophoresis Coupled to Electrospray Ionization Mass Spectrometry
Open this publication in new window or tab >>Electrifying the Molecules of Life: Peptide and Protein Analysis by Capillary Electrophoresis Coupled to Electrospray Ionization Mass Spectrometry
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the current status and novel aspects of the analysis of the molecules of life, i.e. peptides and proteins, using capillary electrophoresis (CE) coupled to mass spectrometry (MS) via (sheathless) electrospray ionization (ESI). Early reports of sheathless CE-ESI-MS were plagued by limited lifetimes of the electrospray emitter. In this thesis, two new approaches, the Black Dust and the Black Jack methods, utilizing polymer-embedded graphite instead of noble metals are presented. These emitters have shown improved long-term stability and proven excellent for sheathless electrospray operation. Failure of an emitter is often caused by electrochemical reactions occurring at the emitter-liquid interface. The electrochemical properties of the graphite coated emitters were therefore evaluated by classical electrochemical methods, such as cyclic voltammetry and chronoamperometry. The graphite coated emitters showed excellent electrochemical stability and properties compared to noble metal and polymer configurations.

Analyte-wall interactions have long been known to cause problems in the CE analysis of biomolecules. This can be circumvented by internal modification of the capillary walls. Additionally, it is of outermost importance to have a stable and sufficiently high electroosmotic flow (EOF) to sustain the electrospray, when using a sheathless approach. New monomer and polymer coatings are presented for rapid and high-efficient CE-ESI-MS separations of peptides and proteins.

Furthermore, the use of CE-ESI coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) shows great potential for rapid proteomic probing of human cerebrospinal fluid. The results are comparable with more established techniques, such as liquid chromatography and two-dimensional gel electrophoresis coupled to MS. However, the CE-ESI-FTICRMS analysis has significantly lower sample consumption and faster analysis time compared to the other techniques. The applications and use of CE-ESI-MS is expected to have a bright future with continued growth as current trends of multidimensional hyphenation and microfabricated devices are further developed and explored.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 44 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 969
Keyword
Analytical chemistry, Capillary electrophoresis (CE), Electrospray ionization (ESI), Mass spectrometry (MS), Peptides, Proteins, Cerebrospinal fluid (CSF), Graphite coating, Monomer coating, Polymer coating, Analytisk kemi
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-4233 (URN)91-554-5947-1 (ISBN)
Public defence
2004-05-19, B:41, BMC, Uppsala, 13:15
Opponent
Supervisors
Available from: 2004-04-20 Created: 2004-04-20 Last updated: 2012-02-08Bibliographically approved

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Wetterhall, MagnusBergquist, Jonas

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