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Miniaturized on-line proteolysis-packed capillary LC-MS for peptide mapping of lactate dehydrogenase
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry, Analytical Chemistry.
In: J Chromatogr AArticle in journal (Refereed) Submitted
URN: urn:nbn:se:uu:diva-90130OAI: oai:DiVA.org:uu-90130DiVA: diva2:162354
Available from: 2003-02-25 Created: 2003-02-25Bibliographically approved
In thesis
1. Development of Micro Liquid Separation Techniques using Electrospray Ionisation Mass Spectrometry in the Analysis of Polar Compounds and Proteins/Peptides
Open this publication in new window or tab >>Development of Micro Liquid Separation Techniques using Electrospray Ionisation Mass Spectrometry in the Analysis of Polar Compounds and Proteins/Peptides
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Electrospray ionisation (ESI) coupled to mass spectrometry (MS) is one of the most important detection techniques for chemical analysis of small drugs as well as large biomolecules in life science today. In this thesis, aspects on improved compatibility between liquid based separation systems and mass spectrometric detection were investigated regarding buffers, sample preparation and analysis of polar compounds as well as peptides and protein digests for enhanced ESI-MS performance.

Capillary electrophoresis (CE) coupled to ESI-MS detection, was evaluated using both a sheath flow interface and a sheathless design. The separation of peptides and small, polar compounds was optimised for both CE-ESI interfaces. The effect of sheath liquid composition was also studied with the aim to improve sensitivity in the ESI-MS detection.

Polar compounds were retained and separated by capillary ion-pair chromatography coupled to ESI-MS detection. Since commonly used ion-pairing reagents are detrimental to the ESI process they were effectively removed before the ionisation by the use of a trapping column after the separation. Alternatively, the ion-pairing reagents were exchanged to volatile constituents.

A method for peptide mapping by liquid chromatography (LC)-ESI-MS was developed for lactate dehydrogenase. The method was further enhanced to involve the proteolysis on-line to the LC-ESI-MS. No manual sample handling was then needed and the total analysis time decreased from 7 to 1.5 hours. The amount of information was also shown to increase in the on-line system.

Finally, the on-line concept was extended to an innovative interface for direct coupling of a pumped liquid flow to an electroosmotically driven flow. This provided a valve-free sample transfer between capillary LC and CE, aiming towards increased peak capacity per unit time for the analysis of complex peptide samples.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 46 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 808
Analytical chemistry, Analytisk kemi
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
urn:nbn:se:uu:diva-3304 (URN)91-554-5534-4 (ISBN)
Public defence
2003-03-21, B21, Uppsala University, Uppsala, 10:15
Available from: 2003-02-25 Created: 2003-02-25 Last updated: 2011-12-09Bibliographically approved

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