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Identification and Characterization of Peptides and Proteins using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Materials Science.
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. , p. 76
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 706
Keywords [en]
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.
Keywords [sv]
Materialvetenskap
National Category
Materials Engineering
Research subject
Molecular Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-1999ISBN: 91-554-5296-5 (print)OAI: oai:DiVA.org:uu-1999DiVA, id: diva2:161609
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
List of papers
1. A 9.4 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: Description and Performance
Open this publication in new window or tab >>A 9.4 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer: Description and Performance
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2000 (English)In: European journal of mass spectrometry, ISSN 1469-0667, E-ISSN 1751-6838, Vol. 6, no 3, p. 267-275Article in journal, Meeting abstract (Refereed) Published
Abstract [en]

9.4 Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers (Bruker BioAPEX-94e) have been installed at the Division of Ion Physics, Uppsala University, and at the Department of Chemistry, University of Warwick, The BioAPEX-94e FT-ICR instrument is built around a high-field, superconducting magnet and a platform with easily interchangeable ion sources [matrix-assisted laser desorption/ionisation (MALDI), secondary ion mass spectrometry (SIMS), electrospray ionisation (ESI) and electron impact/chemical ionisation (EI/CI)I. In this paper a technical description of the instrument is given. Outstanding performance characteristics are demonstrated, notably clear resolution of C59N+ and (C58C2+)-C-13 (mass difference 3.65 mDa) and mass measurement accuracy at the low ppm level. A wide range of applications in Warwick and Uppsala is described, demonstrating the versatility and high performance of the instrument.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-89815 (URN)
Conference
5th European Fourier Transform Mass Spectrometry Workshop, SEP 16-18, 1999, Coventry, England
Available from: 2002-04-23 Created: 2002-04-23 Last updated: 2017-12-14Bibliographically approved
2. Electron Capture Dissociation of Substance-P using a Commercially Available Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
Open this publication in new window or tab >>Electron Capture Dissociation of Substance-P using a Commercially Available Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
1999 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 13, no 6, p. 474-477Article in journal (Refereed) Published
Abstract [en]

Electron capture dissociation of the peptide Substance P is reported for the first time, with an unmodified, commercially available Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The fragmentation pattern is compared with that obtained with collisionally induced dissociation of the ions in the electrospray ion source, and note that electron capture dissociation gives a more easily interpreted spectrum, showing mainly C-fragments. With the exception of the proline residues, which require cleavage of two chemical bonds, we observe all C-fragmental we find the bias voltage of the electron gun not to be very critical.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89816 (URN)10.1002/(SICI)1097-0231(19990330)13:6<474::AID-RCM505>3.0.CO;2-1 (DOI)10204243 (PubMedID)
Available from: 2002-04-23 Created: 2002-04-23 Last updated: 2017-12-14Bibliographically approved
3. 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
4. Analysis of Enzymatically Digested Proteins and Protein Mixtures using a 9.4 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
Open this publication in new window or tab >>Analysis of Enzymatically Digested Proteins and Protein Mixtures using a 9.4 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
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2000 (English)In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 14, no 12, p. 1029-1034Article in journal (Refereed) Published
Abstract [en]

A commercially available 9.4 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was applied in the analysis of tryptic digests of protein mixtures without any separation. First, the method was demonstrated on a mixture of tryptic digests of equine cytochrome c, equine myoglobin and bovine serum albumin. The same method was then applied to human plasma from a healthy blood donor. Computer programs were employed to simplify analysis of the complex spectra. The 2745 peaks in the human plasma electrospray ionization FTICR spectrum could be reduced to 1165 isotopic clusters and 669 unique masses. Out of these, 82 masses matched tryptic fragments of serum albumin with mass measurement errors less than 10 ppm, covering 93% of the sequence. Another 16 masses were assigned to tryptic fragments of transferrin, covering 41% of the sequence on the 10 ppm mass measurement error level (14 within 2 ppm). The mass measurement errors were approximately normal distributed with a standard deviation of 1.7 ppm. This demonstrates the feasibility of combining the ultra-high mass resolving power and accuracy of FTICR mass spectrometry with automated computer analysis for investigating complex biological matrices.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89818 (URN)10.1002/1097-0231(20000630)14:12<1029::AID-RCM984>3.0.CO;2-# (DOI)10861983 (PubMedID)
Available from: 2002-04-23 Created: 2002-04-23 Last updated: 2017-12-14Bibliographically approved
5. Peptide Mapping of Proteins in Human Body Fluids using Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Open this publication in new window or tab >>Peptide Mapping of Proteins in Human Body Fluids using Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
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2002 (English)In: Mass spectrometry reviews (Print), ISSN 0277-7037, E-ISSN 1098-2787, Vol. 21, no 1, p. 2-15Article in journal (Refereed) Published
Abstract [en]

Human body fluids have been rediscovered in the postgenomic era as great sources of biological markers and perhaps particularly as sources of potential protein biomarkers of disease. Analytical tools that allow rapid screening, low sample consumption, and accurate protein identification are of great importance in studies of complex biological samples and clinical diagnosis. Mass spectrometry is today one of the most important analytical tools with applications in a wide variety of fields. One of the fastest growing applications is in proteomics, or the study of protein expression in an organism. Mass spectrometry has been used to find post-translational modifications and to identify key functions of proteins in the human body. In this study, we review the use of human body fluids as sources for clinical markers and present new data that show the ability of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to identify, and characterize proteins in four human body fluids: plasma, cerebrospinal fluid (CSF), saliva, and urine. The body fluids were tryptically digested without any prior separation, purification, or selection, and the digest was introduced into a 9.4 T FTICR mass spectrometer by direct-infusion electrospray ionization (ESI). Even though these samples represent complex biological mixtures, the described method provides information that is comparable with traditional 2D-PAGE data. The sample consumption is extremely low, a few microliters, and the analysis time is only a few minutes. It is, however evident that the separation of proteins and/or peptides must be included in the methodology in order to detect low-abundance proteins and other proteins of biological relevance.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-89819 (URN)10.1002/mas.10016 (DOI)
Available from: 2002-04-23 Created: 2002-04-23 Last updated: 2017-12-14Bibliographically approved
6. Analysis of Tryptically Digested Cerebrospinal Fluid using Capillary Electrophoresis Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Open this publication in new window or tab >>Analysis of Tryptically Digested Cerebrospinal Fluid using Capillary Electrophoresis Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
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In: Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-89820 (URN)
Available from: 2002-04-23 Created: 2002-04-23 Last updated: 2011-03-21
7. Prediction of chromatographic retention and protein identification in liquid chromatography/mass spectrometry
Open this publication in new window or tab >>Prediction of chromatographic retention and protein identification in liquid chromatography/mass spectrometry
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2002 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 74, no 22, p. 5826-5830Article in journal (Refereed) Published
Abstract [en]

Liquid chromatography coupled on- or off-line with mass spectrometry is rapidly advancing as a tool in proteomics capable of dealing with the inherent complexity in biology and complementing conventional approaches based on two-dimensional gel electrophoresis. Proteins can be identified by proteolytic digestion and peptide mass fingerprinting or by searching databases using short-sequence tags generated by tandem mass spectrometry. This paper shows that information on the chromatographic behavior of peptides can assist protein identification by peptide mass fingerprinting in liquid chromatography/mass spectrometry. This additional information is significant and already available at no extra experimental cost.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-89821 (URN)10.1021/ac0256890 (DOI)12463368 (PubMedID)
Available from: 2002-04-23 Created: 2002-04-23 Last updated: 2017-12-14Bibliographically approved
8. Oxidation of Methionine-35 Attenuates Formation of Amyloid β-Peptide 1-40 Oligomers
Open this publication in new window or tab >>Oxidation of Methionine-35 Attenuates Formation of Amyloid β-Peptide 1-40 Oligomers
2002 (English)In: Journal of chemical biology, ISSN 1864-6158, E-ISSN 1864-6166, Vol. 277, p. 19506-19510Article in journal (Refereed) Published
Abstract [en]

Amyloid plaques formed by aggregation of the amyloid β-peptide (Aβ) are an intrinsic component of Alzheimer disease pathogenesis. It has been suggested that oxidation of methionine 35 in Aβ has implications for Alzheimer disease, and it has been shown that oxidation of Met-35 significantly inhibits aggregation in vitro. In this study, the aggregational properties of Aβ-(1–40) before and after Met-35 oxidation were investigated using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The results show that Aβ-(1–40)Met-35(O) trimer and tetramer formation is significantly attenuated as compared with Aβ-(1–40). This suggests that oxidation of Met-35 inhibits a conformational switch in Aβ-(1–40) necessary for trimer but not dimer formation. Random incorporation of Aβ-(1–40) and Aβ-(1–40)Met-35(O) in homo- and heterooligomers could also be observed. This is the first report of an early rate-limiting step in Aβ-(1–40) aggregation. Slowing of the fibrillization process at this early step is likely to support prolonged solubility and clearance of Aβ from brain and may reduce disease progression.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-89822 (URN)10.1074/jbc.M112218200 (DOI)
Available from: 2002-04-23 Created: 2002-04-23 Last updated: 2017-12-14Bibliographically approved
9. Automatic Analysis of Hydrogen/Deuterium Exchange Mass Spectra of Peptides and Proteins using Calculations of Isotopic Distributions
Open this publication in new window or tab >>Automatic Analysis of Hydrogen/Deuterium Exchange Mass Spectra of Peptides and Proteins using Calculations of Isotopic Distributions
2001 (English)In: Journal of the American Society for Mass Spectrometry, ISSN 1044-0305, E-ISSN 1879-1123, Vol. 12, no 11, p. 1153-1162Article in journal (Refereed) Published
Abstract [en]

High mass-resolving power has been shown to be useful for studying the conformational dynamics of proteins by hydrogen/deuterium (H/D) exchange. A computer algorithm was developed that automatically identifies peptides and their extent of deuterium incorporation from H/D exchange mass spectra of enzymatic digests or fragment ions produced by collisionally induced dissociation (CID) or electron capture dissociation (ECD). The computer algorithm compares measured and calculated isotopic distributions and uses a fast calculation of isotopic distributions using the fast Fourier transform (FFT). The algorithm facilitates rapid and automated analysis of H/D exchange mass spectra suitable for high-throughput approaches to the study of peptide and protein structures. The algorithm also makes the identification independent on comparisons with undeuterated control samples. The applicability of the algorithm was demonstrated on simulated isotopic distributions as well as on experimental data, such as Fourier transform ion cyclotron resonance (FTICR) mass spectra of myoglobin peptic digests, and CID and ECD spectra of substance P.

National Category
Natural Sciences Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-89823 (URN)10.1016/S1044-0305(01)00301-4 (DOI)11720389 (PubMedID)
Available from: 2002-04-23 Created: 2002-04-23 Last updated: 2017-12-14Bibliographically approved

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