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Rapid capillary electrophoresis time-of-flight mass spectrometry separations of peptides and proteins using a monoquaternarized piperazine compound (M7C4l) for capillary coatings
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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2008 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 29, no 8, 1619-1625 p.Article in journal (Refereed) Published
Abstract [en]

A monoquaternarized piperazine, 1-(4-iodobutyl) 4-aza-1-azoniabicyclo[2,2,2] octane iodide (M7C4I), has been evaluated as a surface derivatization reagent for CE in combination with TOF MS for the analysis of proteins, peptides, and protein digests. The M7C4I piperazine, at alkaline pH, forms a covalent bond via alkylation of the ionized silanols producing a cationic surface with a highly stable and reversed EOF. The obtained surface yields rapid separations (less than 5 min) of peptides and proteins at acidic pH with high separation efficiencies (up to 1.1 X 10(6) plates/m for peptides and up to 1.8 x 10(6) plates/m for proteins) and no observed bleeding of the coating reagent into the mass spectrometer. The simplicity of the coating procedure also enables fast (2 min) regeneration of the surface, if necessary. This is useful in the analysis of complex samples in order to prevent possible memory effects. The potential of using M7C4I-coated capillaries for MS analysis of complex samples is demonstrated by the separation of peptides, proteins, and protein digests. Even more, the spectacular thing in which large intact proteins with molecular masses over 0.5 MDa could be separated. The coating showed good ability to handle these large proteins with high efficiency and retained peak shape as demonstrated by separation of IgG(1) (150 kDa) and thyroglobulin (669 kDa).

Place, publisher, year, edition, pages
2008. Vol. 29, no 8, 1619-1625 p.
Keyword [en]
Capillary electrophoresis, M7C4l, Peptides, Proteins, Protein digests, Time-of-flight
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-16217DOI: 10.1002/elps.200700737ISI: 000255703100005OAI: oai:DiVA.org:uu-16217DiVA: diva2:43988
Available from: 2008-05-13 Created: 2008-05-13 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Development of Capillary Electrophoresis Methods Coupled to Mass Spectrometry for Biomedical and Pharmaceutical Analysis
Open this publication in new window or tab >>Development of Capillary Electrophoresis Methods Coupled to Mass Spectrometry for Biomedical and Pharmaceutical Analysis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The analysis of large intact proteins and complex biological samples containing drug molecules is a common complicated task for many scientists. However, due to the importance of these molecules, there is a growing interest in pharmaceutical and medicinal research to develop rapid, highly sensitive and efficient analytical techniques. The advantages of capillary electrophoresis (CE) in combination with mass spectrometry (MS) provide a powerful analytical tool. However, further improvement and development of these techniques are required to extend their utility and to meet the challenges of selected analytes. Thus, the scope of this thesis deals with the development of novel analytical methods to achieve efficient and high performance analysis of peptides, intact proteins, digests of complex samples and basic pharmaceutical drug compounds in biological matrices.

Implementation of CE for routine analysis of proteins and complex samples is constrained by the partial adsorption to the capillary wall. Consequently, the use of surface modified capillaries is required to control the surface properties and prevent analyte adsorption. In this thesis, analyte adsorption was successfully prevented using tailored covalent cationic (M7C4I) and electrostatic cationic (PVPy-Me) coatings. Rapid and efficient separations of peptides, proteins and digests of complex samples such as cerebrospinal fluids were obtained with these coatings. The M7C4I coating showed a distinct ability to handle large intact proteins with a molecular size of over 0.5 MDa. The highest peak efficiencies and surprisingly high peak stacking effects were obtained by adding salts to the protein samples. The effect of salt additives on peak efficiencies of intact proteins was further demonstrated and compared using different surface modified capillaries. Additionally, rapid CE-ESI-MS quantification of pharmaceutical drug molecules in human plasma was performed after a SCX-SPE sample preparation method using the M7C4I coating. In conclusion, the results presented in this thesis show the strong potential of CE in combination with MS using electrospray ionization (ESI) for the analysis of peptides and large intact proteins and the applicability for clinical monitoring of the levels of pharmaceutical drug molecules in human plasma with high sensitivity and efficiency.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 804
Keyword
Capillary Electrophoresis, Capillary Surface Modificaions, Electrospray Ionization, Mass Spectrometry, Peptides, Intact Proteins, Basic Pharmaceutical Drug Molecules and Complex Biological Samples
National Category
Other Basic Medicine
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-143814 (URN)978-91-554-7996-1 (ISBN)
Public defence
2011-03-11, C8:301, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Note
Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 734Available from: 2011-02-18 Created: 2011-01-25 Last updated: 2011-04-04Bibliographically approved
2. Quantitative Bioanalysis: Liquid separations coupled to targeted mass spectrometric measurements of bioactive compounds
Open this publication in new window or tab >>Quantitative Bioanalysis: Liquid separations coupled to targeted mass spectrometric measurements of bioactive compounds
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Performing quantitative analysis of targeted bioactive compounds in biological samples, such as blood plasma, cerebrospinal fluid or extracts from pig liver, put high demands on the ruggedness of the method acquiring the results. In addition to the complexity of the sample matrix, the bioactive compounds targeted for analysis usually have low levels of natural abundance, further increasing the demand on the analytical method sensitivity. Furthermore, quantitation of analytes at trace levels in the presence of large amounts of interfering species in biofluids must aim for repeatable precision, high accuracy ensuring the closeness to the true values, a linear response spanning over several orders of magnitude and low limits of quantitation to be valid for monitoring disease states in clinical analysis.

An analytical method most commonly follow a certain order of events, called the analytical chain, which includes; experimental planning, sampling, sample pre-treatment, separation of species, detection, evaluation, interpretation and validation, all equally important for the outcome of the results.

In this thesis, the scope has been to create novel methods, or to refine already existing methods, in order to achieve even better performances of the different events in the analytical chain.

One of the aspects has been to sample and enrich analytes in vivo by the use of solid supported microdialysis, giving the advantage of almost real-time monitoring of analyte levels within a living host with targeted selectivity due to the analyte affinity for solid particles. Another aspect to selectively clean and enrich analytes in a complex matrix has been developed and automated on-line by the use of a column-switching technique before the analytical separation. By using several steps of extraction and separation coupled on-line to selected detection by the use of a triple quadrupole mass spectrometer facilitates great selectivity of species. The mass spectrometer also gives the ability to distinguish between isotopically labelled analogues coeluting with the analytes yielding the necessary accuracy for quantitative evaluation.

Both development of analytical methods and clinical applications has been performed, as well as improvements of existing techniques, all to improve the quantitation of trace levels of targeted analytes in biofluids.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 412
Keyword
Analytical chemistry, Method development, Enhanced microdialysis, Column switching, Liquid chromatography, Capillary electrophoresis, Electrospray ionization, Mass spectrometry, Triple quadrupole, Time-of-flight, Quantitation, Validation, Analytisk kemi
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-8581 (URN)978-91-554-7135-4 (ISBN)
Public defence
2008-04-10, B22, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2008-03-17 Created: 2008-03-17 Last updated: 2013-06-20Bibliographically approved

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Elhamili, AnisaWetterhall, MagnusArvidsson, BjörnBergquist, Jonas

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