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Quantitative Analysis of Tryptic Protein Mixtures Using Electrospray Ionization Fourier Transform Ion Cyclotron Mass Spectrometry
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
2004 In: Journal of Proteome Research, ISSN 1535-3893, Vol. 3, no 3, 587-594 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2004. Vol. 3, no 3, 587-594 p.
Identifiers
URN: urn:nbn:se:uu:diva-92812OAI: oai:DiVA.org:uu-92812DiVA: diva2:166111
Available from: 2005-04-07 Created: 2005-04-07 Last updated: 2014-10-02
In thesis
1. Analysis of Complex Biological Samples using Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Open this publication in new window or tab >>Analysis of Complex Biological Samples using Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Studies of protein and peptide expression are vital in order to understand complex biological systems. As demonstrated in this thesis, on-line packed capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS) is a useful analytical tool for such studies.

A proteomics method, based on global tryptic digestion and subsequent separation and detection of the peptides by LC-FTICR MS, was developed for qualitative analysis of body fluids. Initial experiments on cerebrospinal fluid (CSF) provided results that were comparable or superior to those achieved by more time- and sample-consuming techniques. The method was also successfully applied on plasma and amniotic fluid. One of the major challenges in proteomics is the broad dynamic range of proteins in biological matrices. The advantages of removing high-abundant components from CSF and plasma prior to MS were demonstrated.

In order to search for potential biomarkers, mass chromatograms of CSF from patients suffering from amyotrophic lateral sclerosis (ALS) and controls were compared using an in-house constructed pattern recognition program. ALS-specific patterns were observed, and four out of five unknown samples were correctly assigned. Alternative strategies to quantitatively compare two pools of samples rely on differential chemical labeling. The performance of one such method, quantification-using-enhanced-signal-tags, was investigated in complex sample analysis. The experimental intensity ratios were proven to be consistent with the prepared concentration ratios of abundant proteins in CSF.

Finally, the thesis reports on the first experiments where electron capture dissociation (ECD) was successfully incorporated in on-line LC-MS experiments. ECD and nozzle-skimmer fragmentation were applied to a sample of endocrine peptides extracted from mouse pancreatic islets. The two fragmentation methods provided complementary information. However, the method needs further optimization before it can be applied in the analysis of more complex samples, such as body fluids.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 31
Keyword
Analytical chemistry, mass spectrometry, liquid chromatography, protein, proteomics, peptide, Fourier transform ion cyclotron resonance mass spectrometry, cerebrospinal fluid, amyotrophic lateral sclerosis, electrospray ionization, electron capture dissociation, Analytisk kemi
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-5729 (URN)91-554-6198-0 (ISBN)
Public defence
2005-05-04, Room B22, BMC, Husargatan 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2005-04-07 Created: 2005-04-07 Last updated: 2013-03-22Bibliographically approved

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