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Interfacing Complementary Separation Techniques with Mass Spectrometry Utilizing Electrophoresis, Nanoparticles, and Functionalized Magnetic Beads
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
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: urn:nbn:se:uu:diva-183666ISBN: 978-91-554-8529-0 (print)OAI: oai:DiVA.org:uu-183666DiVA: diva2:563717
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
List of papers
1. Efforts to improve detection sensitivity for capillary electrophoresis coupled to atmospheric pressure photoionization mass spectrometry.
Open this publication in new window or tab >>Efforts to improve detection sensitivity for capillary electrophoresis coupled to atmospheric pressure photoionization mass spectrometry.
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2010 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 24, no 9, 1260-1264 p.Article in journal (Refereed) Published
Abstract [en]

Electrospray ionization performs best with volatile buffers. However, generally the best separation performance for capillary electrophoresis (CE) is achieved with non-volatile buffers. Hyphenation of CE with mass spectrometry (MS) utilizing atmospheric pressure photoionization (APPI) enables use of a wider range of separation buffers without compromising detection sensitivity. As APPI is considered to be mass flow sensitive, the use of a larger inner diameter separation capillary (75 microm) allows larger volumes to be injected, without decreased separation performance, thus providing improved sensitivity (approx. a factor of 10), compared to the use of a 25 microm capillary. However, nebulizing gas flow and position of capillary tip in the sprayer have to be carefully optimized to prevent excessive band broadening. Further improvement in sensitivity (approx. a factor of 2) was obtained by decreasing the distance between the sprayer and ionization region, indicating that a specially designed CE/APPI-MS interface for low flow rates will be favourable.

Place, publisher, year, edition, pages
John Wiley & Sons Ltd, 2010
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-122426 (URN)10.1002/rcm.4510 (DOI)000277352700006 ()20391596 (PubMedID)
Available from: 2010-04-12 Created: 2010-04-12 Last updated: 2017-12-12Bibliographically approved
2. Continuous full filling capillary electrochromatography-electrospraying chromatographic nanoparticles
Open this publication in new window or tab >>Continuous full filling capillary electrochromatography-electrospraying chromatographic nanoparticles
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2011 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 32, no 2, 261-267 p.Article in journal (Refereed) Published
Abstract [en]

The influence of instrumental parameters affecting the ionization in continuous full filling capillary electrochromatography/electrospray ionization mass spectrometry (CFF-CEC/ESI-MS) was investigated. The investigated parameters were the BGE and sheath liquid ion strength and organic modifier content, the nebulizer gas pressure, and the concentration of nanoparticles in the BGE. It was found that the nebulizer pressure had the largest influence on the separation efficiency and apparent retention. It was shown that even the lowest pressure investigated was sufficient to guide the nanoparticle flow away from the mass spectrometer inlet. A nebulizer pressure of 5 psi was found to be optimal; increasing the pressure significantly decreased the separation efficiency due to the generation of a hydrodynamic flow. Generally, the ion strength of both the BGE and the sheath liquid were found to have very moderate effects on the separation of a homologous series of dialkyl phthalates, whereas the ionization efficiency was found to be unaffected by the nanoparticles and the separation efficiency was found to increase with increasing concentrations up to 3.8 mg/mL, whereafter it was observed to drop. The optimized method was linear over a wide concentration range and presented LOD and LOQ more than threefold lower than those previously reported using CFF-CEC/ESI-MS.

Keyword
Continuous full filling capillary electrochromatography, Nanoparticles, Nebulizing gas pressure, Pseudostationary phase, Sheath liquid
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-145492 (URN)10.1002/elps.201000261 (DOI)000287156700009 ()21254124 (PubMedID)
Funder
Swedish Research Council
Available from: 2011-02-09 Created: 2011-02-09 Last updated: 2017-12-11Bibliographically approved
3. One step solution for high molecular weight protein depletion and selective enrichment of low molecular weight proteins using functionalized magnetic beads
Open this publication in new window or tab >>One step solution for high molecular weight protein depletion and selective enrichment of low molecular weight proteins using functionalized magnetic beads
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(English)Manuscript (preprint) (Other academic)
Keyword
Magnetic Beads, Functionalized, High molecular weight proteins, Low molecular weight proteins, Electrophoresis, Enrichment, Plasma
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-183588 (URN)
Available from: 2012-10-29 Created: 2012-10-29 Last updated: 2013-01-02
4. 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
5. Capillary Electrophoresis-Mass Spectrometry (CZE-MS) Analysis of Intact Monoclonal Antibodies using bare fused silica
Open this publication in new window or tab >>Capillary Electrophoresis-Mass Spectrometry (CZE-MS) Analysis of Intact Monoclonal Antibodies using bare fused silica
(English)Manuscript (preprint) (Other academic)
National Category
Analytical Chemistry Other Basic Medicine
Identifiers
urn:nbn:se:uu:diva-183590 (URN)
Available from: 2012-10-29 Created: 2012-10-29 Last updated: 2018-01-12

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