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Analysis of Complex Biological Samples using Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
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. , p. 62
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 31
Keywords [en]
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
Keywords [sv]
Analytisk kemi
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-5729ISBN: 91-554-6198-0 (print)OAI: oai:DiVA.org:uu-5729DiVA, id: diva2:166115
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
List of papers
1. Protein identification in cerebrospinal fluid using packed capillary liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry
Open this publication in new window or tab >>Protein identification in cerebrospinal fluid using packed capillary liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry
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2003 In: Proteomics, ISSN 1615-9853, Vol. 3, no 2, p. 184-190Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-92808 (URN)
Available from: 2005-04-07 Created: 2005-04-07 Last updated: 2011-03-21
2. Cerebrospinal fluid protein patterns in neurodegenerative disease revealed by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry
Open this publication in new window or tab >>Cerebrospinal fluid protein patterns in neurodegenerative disease revealed by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry
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2004 In: Proteomics, Vol. 4, no 12, p. 4010-4018Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-92809 (URN)
Available from: 2005-04-07 Created: 2005-04-07 Last updated: 2011-03-21
3. An Explorative Study of the Protein Composition of Amniotic Fluid by Liquid Chromatography Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Open this publication in new window or tab >>An Explorative Study of the Protein Composition of Amniotic Fluid by Liquid Chromatography Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
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2004 In: Journal of Proteome Research, ISSN 1535-3893, Vol. 3, no 4, p. 884-889Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-92810 (URN)
Available from: 2005-04-07 Created: 2005-04-07 Last updated: 2011-03-21
4. Depletion of High-Abundant Proteins in Body Fluids Prior to Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Open this publication in new window or tab >>Depletion of High-Abundant Proteins in Body Fluids Prior to Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
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2005 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 2, p. 410-416Article in journal (Refereed) Published
Abstract [en]

Today, proteomics is an exciting approach to discover potential biomarkers of different disorders. One challenge with proteomics experiments is the wide concentration range of proteins in various tissues and body fluids. The most abundant component in human body fluids, human serum albumin (HSA), is present at concentrations corresponding to approximately 50% of the total protein content in e.g., plasma and cerebrospinal fluid (CSF). If this component could be selectively removed, then the chances of observing lower-abundance component of clinical interest would be greatly improved. There are today several approaches of varying specificity available for depletion. In this study, the properties of two commercially available kits, for the removal of HSA and HSA and immunoglobulin G (lgG), respectively, were compared, and the benefits of using depletion steps prior to on-line LC-FTICR MS were evaluated. Both methods were applied on plasma and CSF. To our knowledge, these are the first results reported for CSF. Also, the combination with electrospray LC-FTICR MS is novel. The proportion of depleted HSA and lgG was estimated using global labeling markers for peptide quantification. Both depletion-methods provided a significant reduction of HSA, and the identification of lower abundant components was clearly facilitated. A higher proportion of HSA was removed using the affinity-based removal kit, and consequently more proteins could be identified using this approach.

Keywords
FTICR MS, LC, HSA, plasma, CSF, protein, QUEST-markers
National Category
Physical Sciences
Identifiers
urn:nbn:se:uu:diva-92541 (URN)10.1021/pr049812a (DOI)15822917 (PubMedID)
Available from: 2005-01-14 Created: 2005-01-14 Last updated: 2017-12-14
5. Quantitative Analysis of Tryptic Protein Mixtures Using Electrospray Ionization Fourier Transform Ion Cyclotron Mass Spectrometry
Open this publication in new window or tab >>Quantitative Analysis of Tryptic Protein Mixtures Using Electrospray Ionization Fourier Transform Ion Cyclotron Mass Spectrometry
2004 In: Journal of Proteome Research, ISSN 1535-3893, Vol. 3, no 3, p. 587-594Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-92812 (URN)
Available from: 2005-04-07 Created: 2005-04-07 Last updated: 2014-10-02
6. Reproducibility of Tryptic Digestion Investigated by Quantitative Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Open this publication in new window or tab >>Reproducibility of Tryptic Digestion Investigated by Quantitative Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
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2005 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 2, p. 394-399Article in journal (Refereed) Published
Abstract [en]

In this study, the reproducibility of tryptic digestion of complex solutions was investigated using liquid chromatography Fourier transform ion cyclotron resonance (LC FT-ICR) mass spectrometry. Tryptic peptides, from human cerebrospinal fluid, (CSF) was labeled with Quantification-Using-Enhanced-Signal-Tags (QUEST)-markers, or 1-((H4)nicotinoyloxy)- and 1-((D4)nicotinoyloxy)-succinimide ester markers. The analysis was performed on abundant proteins with respect-to-intensity ratios and sequence coverage and obtained by comparing differently labeled components from one or different pools. To interpret the dynamics in the proteome, one must be able to estimate the error introduced in each experimental steps. The intra sample variation due to derivatization was approximately 10%. The inter sample variation depending on derivatization and tryptic digestion was not more than approximately 30%. These experimental observations provide a range for the up- and down-regulations that are possible to study with electrospray ionization LC FT-ICR mass spectrometry.

Keywords
cerebrospinal fluid, LC FT-ICR mass spectrometry, QUEST-markers, quantification, tryptic reproducibility, 1-((H4)nicotinoyloxy)- and 1-((D4)nicotinoyloxy-succinimide ester markers
National Category
Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-92540 (URN)10.1021/pr049809r (DOI)15822915 (PubMedID)
Available from: 2005-01-14 Created: 2005-01-14 Last updated: 2017-12-14
7. Liquid Chromatography and Electron Capture Dissociation in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Open this publication in new window or tab >>Liquid Chromatography and Electron Capture Dissociation in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
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2002 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 16, no 10, p. 988-992Article in journal (Refereed) Published
Abstract [en]

Liquid separation methods in combination with electrospray mass spectrometry as well as the recently introduced fragmentation method electron capture dissociation (ECD) have become powerful tools in proteomics research. This paper presents the results of the first successful attempts to combine liquid chromatography (LC) and Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) with ECD in the analysis of a mixture of standard peptides and of a bovine serum albumin tryptic digest. A novel electron injection system provided conditions for ECD sufficient to yield extensive sequence information for the most abundant peptides in the mixtures on the time-scale of the chromatographic separation. The results suggest that LC/ECD-FTICRMS can be employed in the characterization of peptides in enzymatic digests of proteins or protein mixtures and identify and localize posttranslational modifications.

National Category
Analytical Chemistry Subatomic Physics
Identifiers
urn:nbn:se:uu:diva-89817 (URN)10.1002/rcm.667 (DOI)000175539300014 ()
Available from: 2002-04-23 Created: 2002-04-23 Last updated: 2017-12-14
8. A novel mass spectrometric approach to the analysis of hormonal peptides in extracts of mouse pancreatic islets
Open this publication in new window or tab >>A novel mass spectrometric approach to the analysis of hormonal peptides in extracts of mouse pancreatic islets
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2003 In: European Journal of Biochemistry, ISSN 0014-2956, Vol. 270, no 15, p. 3146-3152Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-92815 (URN)
Available from: 2005-04-07 Created: 2005-04-07 Last updated: 2011-03-21

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