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A Site-Specific Investigation with Hydrogen/Deuterium Exchange Using Collision-Induced Dissociation and Electron Capture Dissociation
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Technology, Department of Engineering Sciences.
Manuscript (Other academic)
URN: urn:nbn:se:uu:diva-92545OAI: oai:DiVA.org:uu-92545DiVA: diva2:165667
Available from: 2005-01-14 Created: 2005-01-14 Last updated: 2010-01-13Bibliographically approved
In thesis
1. Method Development in Quantitative and Structural Proteomics using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Open this publication in new window or tab >>Method Development in Quantitative and Structural Proteomics using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, methods for studying different aspects of proteomics were developed with Fourier Transform Ion Cyclotron Resonance, (FTICR), mass spectrometry. The FTICR technique provides ultra-high mass resolving power, mass accuracy at sub ppm level and sensitivity in the attomole region.

Methods for quantifying biomarkers in body fluids such as cerebrospinal fluid, (CSF), and plasma were developed. Two sets of global markers with different properties were used for quantitative analysis; S-Methyl Thioacetimidate, (SMTA), and S-Methyl Thiopropionimidate, (SMTP), and [H4]- and [D4]-1-Nicotinoyloxy succinimide ester. Reduced ion suppression and higher sensitivity was obtained by coupling a High Performance Liquid Chromatography, (HPLC), system to the FTICR mass spectrometer.

In body fluids, proteins and peptides are present in a broad dynamic concentration range. Therefore, depleting abundant proteins prior to analysis results in decreased ion suppression and increased sensitivity. Two commercial depletion kits were evaluated with the SMTA- and SMTP-markers.

For both types of global markers, the experimental error for quantitative analysis of abundant proteins was less than 30%. This provides a lower limit for the protein up- and down regulations in complex solutions that can be monitored with HPLC-FTICR mass spectrometry.

Together with the identity and quantity of selected proteins the structure, dynamics and interactions with other molecules are of great importance. The later can be elucidated with Hydrogen/Deuterium Exchange, (HDX), mass spectrometry. Structural information at high resolution can be obtained with Collision-Induced Dissociation, (CID), HDX mass spectrometry. In this thesis, exchange rates of amide hydrogens in peptides were in excellent agreement with NMR results.

In some cases, the CID-fragments have different gas-phase exchange properties and as a consequence the solution phase exchange process can not be monitored. By applying Electron Capture Dissociation, (ECD), at ultra-high vacuum, the exchange process at a specific residue could be monitored.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 52 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 6
Biotechnology, Cerebrospinal Fluid, Electrospray Ionization, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Global Markers, High Performance Liquid Chromatography, Gas-Phase Exchange, Hydrogen/Deuterium Exchange, Plasma, Protoemics, Quantification, Structural Elucidation, Bioteknik
National Category
Industrial Biotechnology
urn:nbn:se:uu:diva-4761 (URN)91-554-6134-4 (ISBN)
Public defence
2005-02-04, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 13:00
Available from: 2005-01-14 Created: 2005-01-14Bibliographically approved

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