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A polyamine coating for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry of proteins and peptides.
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, Analytical 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, Analytical Chemistry.
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2004 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 25, no 13, 2090-2099 p.Article in journal (Refereed) Published
Abstract [en]

A procedure for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) of proteins is presented. The use of a newly presented capillary coating, PolyE-323, provided fast separations of typically a few minutes with high efficiency, good deactivation, and no bleeding into the mass spectrometer. Capillaries coated with PolyE-323 showed high stability over a range of pH 2-10, and tolerance towards methanol and acetonitrile, two modifiers commonly used in CE-ESI-MS. Due to the speed and simplicity of the coating procedure, the polymeric surface could, if necessary, easily be regenerated. This capability is especially valuable when working with samples of complex matrix, where a capillary surface cleaning step might be desired in order to eliminate possible memory effects. The potential of PolyE-323-coated capillaries in bioanalysis using CE-ESI-MS was demonstrated by analyzing peptides and proteins up to 66 kDa using time of flight (TOF)-MS. Due to the stable, anodal electroosmotic flow generated by the coating, the use of a sheathless ESI interface was enabled, demonstrated in peptide analysis with attomole sensitivity. The fast on-line CE-ESI-TOF system using PolyE-323-coated capillaries provided efficient separation and detection of a large number of peaks in a short time, exemplified by the analysis of a tryptic digest of bovine serum albumin (BSA). The capability of the developed capillary surface coating was demonstrated by the separation of human plasma and cerebrospinal fluid (CSF).

Place, publisher, year, edition, pages
2004. Vol. 25, no 13, 2090-2099 p.
Keyword [en]
Capillary electrophoresis, mass spectrometry, peptides, polyamine coating, proteins
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-98065DOI: 10.1002/elps.200305787ISI: 000222890600019PubMedID: 15237410OAI: oai:DiVA.org:uu-98065DiVA: diva2:173235
Available from: 2009-02-20 Created: 2009-02-06 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Surface Modified Capillaries in Capillary Electrophoresis Coupled to Mass Spectrometry: Method Development and Exploration of the Potential of Capillary Electrophoresis as a Proteomic Tool
Open this publication in new window or tab >>Surface Modified Capillaries in Capillary Electrophoresis Coupled to Mass Spectrometry: Method Development and Exploration of the Potential of Capillary Electrophoresis as a Proteomic Tool
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The increased knowledge about the complexity of the physiological processes increases the demand on the analytical techniques employed to explore them. A comprehensive analysis of the entire sample content is today the most common approach to investigate the molecular interplay behind a physiological deviation. For this purpose a method that offers a number of important properties, such as speed and simplicity, high resolution and sensitivity, minimal sample volume requirements, cost efficiency and robustness, possibility of automation, high-throughput and wide application range of analysis is requested. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has a great potential and fulfils many of these criteria. However, further developments and improvements of these techniques and their combination are required to meet the challenges of complex biological samples.

Protein analysis using CE is a challenging task due to protein adsorption to the negatively charged fused-silica capillary wall. This is especially emphasised with increased basicity and size of proteins and peptides. In this thesis, the adsorption problem was addressed by using an in-house developed physically adsorbed polyamine coating, named PolyE-323. The coating procedure is fast and simple that generates a coating stable over a wide pH range, 2-11. By coupling PolyE-323 modified capillaries to MS, either using electrospray ionisation (ESI) or matrix-assisted laser desorption/ionisation (MALDI), successful analysis of peptides, proteins and complex samples, such as protein digests and crude human body fluids were obtained. The possibilities of using CE-MALDI-MS/MS as a proteomic tool, combined with a proper sample preparation, are further demonstrated by applying high-abundant protein depletion in combination with a peptide derivatisation step or isoelectric focusing (IEF). These approaches were applied in profiling of the proteomes of human cerebrospinal fluid (CSF) and human follicular fluid (hFF), respectively. Finally, a multiplexed quantitative proteomic analysis was performed on a set of ventricular cerebrospinal fluid (vCSF) samples from a patient with traumatic brain injury (TBI) to follow relative changes in protein patterns during the recovery process.

The results presented in this thesis confirm the potential of CE, in combination with MS, as a valuable choice in the analysis of complex biological samples and clinical applications.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 603
Keyword
Topology optimization, Design optimization, Material distribution, Wave propagation problems, Inverse problems, Acoustic devices, Medical microwave tomography, High performance computing
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-9554 (URN)978-91-554-7411-9 (ISBN)
Public defence
2009-03-20, B41, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2009-02-20 Created: 2009-02-06 Last updated: 2009-03-25Bibliographically approved
2. Tailormade Surfaces for Extended CE Applications
Open this publication in new window or tab >>Tailormade Surfaces for Extended CE Applications
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The combination of capillary electrophoresis (CE) and mass spectrometry (MS) constitutes a powerful microanalytical system in the fields of biology, medicine and chemistry. This thesis describes the development of three novel capillary coatings and demonstrates how these extend the utility of CE as a high-efficiency separation technique in protein analysis and biopharmaceutical drug screening.

Due to the rapidly growing interest in characterizing the human proteome, there is an increased need for rapid protein separations. The use of CE in protein analysis is, however, nontrivial due to problems with protein adsorption to the fused-silica capillary walls. In this thesis, this problem was addressed by developing two novel, physically adsorbed, cationic polymer surface coatings, denoted PolyE-323 and Q-agarose. By using simple rinsing protocols, highly reproducible coatings, stable over a wide range of pH 2-11 were generated. Successful protein separations using cationic-coated capillaries in CE-MS, equipped with either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), has been demonstrated.

In the pharmaceutical industry, favorable pharmacokinetic properties of a candidate drug, such as high bioavailability after oral administration, are crucial for a high success rate in clinical development. Tools for prediction of biopharmaceutically relevant drug properties are important in order to identify and discard poor candidate drugs as soon as possible. In this thesis, a membrane mimetic coating was developed by electrostatically immobilizing liposomes to the capillary wall, via an anchoring sublayer of Q-agarose. The liposome-coated capillaries were demonstrated in on-line CE-MS for prediction of drug membrane permeability.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 50 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 962
Keyword
Analytical chemistry, capillary electrophoresis, mass spectrometry, surface properties, coatings, polymers, liposomes, proteins, drugs, Analytisk kemi
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-4217 (URN)91-554-5934-X (ISBN)
Public defence
2004-05-14, B42, BMC, Husargatan 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2004-04-23 Created: 2004-04-23 Last updated: 2012-02-08Bibliographically approved
3. Development of Liquid-based Separation Techniques using Tailored Surfaces for Analysis of Biological Samples
Open this publication in new window or tab >>Development of Liquid-based Separation Techniques using Tailored Surfaces for Analysis of Biological Samples
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Development and improvement of analytical techniques are vital in analytical chemistry research. This thesis describes the development and use of tailored surfaces for bioanalytical applications.

In sample preparation, solid phase extraction is often used and the development of a protocol for extraction on a molecular imprinted polymer (MISPE) directly from plasma sample is presented. Molecular imprinted polymers (MIP) offer selective sorbents for the imprinted analyte. MISPE has mainly been used in organic phase but in this thesis the development of a protocol for direct extraction of the analyte form an aqueous phase is described.

For analysis of complex samples a separation step is often needed. The growing interest in analysis of biological samples and analysis of the human proteome and potential biomarkers has increased the interest in developing new separation techniques. Capillary electrophoresis (CE) has evolved into an important technique for use in analysis of body fluids. In this thesis a novel polyamine coating named PolyE 323 tailored for minimizing the adsorption of basic proteins to the surface is introduced. A straightforward coating protocol, with four simple rinsing steps, was developed. The coating was highly reproducible and useable over a wide pH range. Successful protein separations on PolyE-323-coated capillaries coupled to electrospray ionization mass spectrometry (ESI-MS) were demonstrated.

The coated capillaries were also used in studies of protein content of aqueous humor samples from cataract patients as a complement to capillary liquid chromatography. In the studies presented the protein content of aqueous humor samples from two clinical groups was compared. By using capillary liquid separation techniques coupled to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and MS/MS in combination with isobaric tags for relative and absolute quantitation (iTRAQ) the identity and relative concentrations of proteins in the samples were evaluated. Earlier studies of the proteins in these kinds of samples have mainly been done with techniques using immunological detection where the proteins of interest were chosen in advance. In this thesis it was shown that liquid-based separation techniques are a good complement and by using the mass spectrometry approach presented the protein content of the samples could be evaluated without bias.

Place, publisher, year, edition, pages
Uppsala: Universitetsbiblioteket, 2008. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 577
Keyword
aqueous humor, biological samples, capillary electrophoresis, coatings, drugs, liquid chromatography, molecular imprinted polymers, peptides, proteins, pseudoexfoliation
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-9399 (URN)978-91-554-7350-1 (ISBN)
Public defence
2008-12-11, B42, BMC, Husargatan 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2008-11-20 Created: 2008-11-20 Last updated: 2012-02-08Bibliographically approved
4. 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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