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Liquid Chromatography and Electron Capture Dissociation in Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science.
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry, Analytical Chemistry.
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2002 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 16, no 10, 988-992 p.Article 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.

Place, publisher, year, edition, pages
2002. Vol. 16, no 10, 988-992 p.
National Category
Analytical Chemistry Subatomic Physics
Identifiers
URN: urn:nbn:se:uu:diva-89817DOI: 10.1002/rcm.667ISI: 000175539300014OAI: oai:DiVA.org:uu-89817DiVA: diva2:161602
Available from: 2002-04-23 Created: 2002-04-23 Last updated: 2017-12-14
In thesis
1. Identification and Characterization of Peptides and Proteins using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Open this publication in new window or tab >>Identification and Characterization of Peptides and Proteins using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mass spectrometry has in recent years been established as the standard method for protein identification and characterization in proteomics with excellent intrinsic sensitivity and specificity. Fourier transform ion cyclotron resonance is the mass spectrometric technique that provides the highest resolving power and mass accuracy, increasing the amount of information that can be obtained from complex samples. This thesis concerns how useful information on proteins of interest can be extracted from mass spectrometric data on different levels of protein structure and how to obtain this data experimentally. It was shown that it is possible to analyze complex mixtures of protein tryptic digests by direct infusion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and identify abundant proteins by peptide mass fingerprinting. Coupling on-line methods such as liquid chromatography and capillary electrophoresis increased the number of proteins that could be identified in human body fluids. Protein identification was also improved by novel statistical methods utilizing prediction of chromatographic behavior and the non-randomness of enzymatic digestion. To identify proteins by short sequence tags, electron capture dissociation was implemented, improved and finally coupled on-line to liquid chromatography for the first time. The combined techniques can be used to sequence large proteins de novo or to localize and characterize any labile post-translational modification. New computer algorithms for the automated analysis of isotope exchange mass spectra were developed to facilitate the study of protein structural dynamics. The non-covalent interaction between HIV-inhibitory peptides and the oligomerization of amyloid β-peptides were investigated, reporting several new findings with possible relevance for development of anti-HIV drug therapies and understanding of fundamental mechanisms in Alzheimer’s disease.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 76 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 706
Keyword
Materials science, Peptide, protein, peptide mass fingerprinting, identification, sequencing, protein structure, non-covalent interaction, modification, electrospray ionization, liquid chromatography, capillary electrophoresis, electron capture dissociation, Fourier transform ion cyclotron resonance mass spectrometry, cerebrospinal fluid, amyloid β-peptide, Alzheimer’s disease., Materialvetenskap
National Category
Materials Engineering
Research subject
Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-1999 (URN)91-554-5296-5 (ISBN)
Public defence
2002-05-17, Siegbahnsalen, Uppsala, 10:00 (English)
Opponent
Available from: 2002-04-23 Created: 2002-04-23 Last updated: 2010-01-14Bibliographically approved
2. 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
3. High Rate Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Open this publication in new window or tab >>High Rate Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Snabb fragmenteringsmetod genom elektroninfågning i Fouriertransform-joncyklotronresonans-masspektrometri
Abstract [en]

Advances in science and technology during the past decade have greatly enhanced the level of the structural investigation of macromolecules – peptides and proteins. Biological mass spectrometry has become one of the most precise and sensitive techniques in peptide and protein analysis. However, increasing demands of biotechnological applications require further progress to be made.

In the present thesis the development and improvement of peptide and protein characterization methods and techniques based on ion-electron and ion-photon reactions in electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry are described. The focus is on the development of the electron capture dissociation method, recently discovered by the group of professor McLafferty, into a high rate, efficient tandem mass spectrometrical technique.

The rate and reliability of the electron capture dissociation technique were greatly increased by implementation of low-energy pencil electron beam injection systems based on indirectly heated dispenser cathodes. Further implementation of a hollow electron beam injection system combined, in a single experimental configuration, two rapid fragmentation techniques, high rate electron capture dissociation and infrared multiphoton dissociation. Simultaneous and consecutive irradiations of trapped ions with electrons and photons extended the possibilities for ion activation/dissociation reaction schemes and lead to improved peptide and protein characterization. Using these improvements, high rate electron capture dissociation was employed in time-limited experiments, such as liquid chromatography–tandem mass spectrometry and capillary electrophoresis-tandem mass spectrometry.

The analytical applications of the developed techniques have been demonstrated in top-down sequencing of peptides and proteins up to 29 kDa, improved sequencing of peptides with multiple disulfide bridges and secondary fragmentation (w-ion formation), as well as extended characterization of peptide mixtures separated by liquid chromatography and capillary electrophoresis. For instance, the dissociation of peptides resulting from enzymatic digestion of proteins provided complementary structural information on peptides and proteins, as well as their post-translational modifications.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 66 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 956
Keyword
Physics, mass spectrometry, Fourier transform ion cyclotron resonance, peptides and proteins, ion-electron interaction, electron capture dissociation, infrared multiphoton dissociation, liquid chromatography, capillary electrophoresis, Fysik
National Category
Physical Sciences
Identifiers
urn:nbn:se:uu:diva-4136 (URN)91-554-5917-X (ISBN)
Public defence
2004-04-23, Häggsalen, Ångström Laboratory, Lagerhydsvagen 1, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2004-03-25 Created: 2004-03-25 Last updated: 2011-03-21Bibliographically approved

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Ramström, MargaretaBergquist, Jonas

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