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Depletion of High-Abundant Proteins in Body Fluids Prior to Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Ion Physics.
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2005 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 4, no 2, 410-416 p.Article 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.

Place, publisher, year, edition, pages
2005. Vol. 4, no 2, 410-416 p.
Keyword [en]
FTICR MS, LC, HSA, plasma, CSF, protein, QUEST-markers
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-92541DOI: 10.1021/pr049812aPubMedID: 15822917OAI: oai:DiVA.org:uu-92541DiVA: diva2:165663
Available from: 2005-01-14 Created: 2005-01-14 Last updated: 2014-09-17
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 6
Keyword
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
Identifiers
urn:nbn:se:uu:diva-4761 (URN)91-554-6134-4 (ISBN)
Public defence
2005-02-04, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 13:00
Opponent
Supervisors
Available from: 2005-01-14 Created: 2005-01-14Bibliographically 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

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Ramström, MargaretaBergquist, Jonas

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