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Proteome profiling of human cerebrospinal fluid: exploring the potential of capillary electrophoresis with surface modified capillaries for analysis of complex biological samples.
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, Chemistry, Department of Physical and Analytical Chemistry.
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, Chemistry, Department of Physical and Analytical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
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, Chemistry, Department of Physical and Analytical Chemistry.
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2008 (English)In: European journal of mass spectrometry, ISSN 1469-0667, Vol. 14, no 4, 249-260 p.Article in journal (Refereed) Published
Abstract [en]

A bottom-up proteomic approach, based on capillary electrophoresis (CE) in combination with matrix- assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToF MS), was used to analyze immunoaffinity depleted human cerebrospinal fluid (CSF) and compare it with a non-depleted sample. After enzymatic digestion and desalting, the tryptic peptides were separated by CE using PolyE-323 modified capillaries and fractionated off-line onto MALDI target plates for further analysis by MALDI-MS and MS/MS. The protein profile of the depleted sample was compared with non depleted CSF. Overall, 85 proteins were identified with 95% significance in both samples. The significance scores for proposed biomarkers, such as amyloid-like protein 1 precursor, could be increased up to 12 times after the depletion. Other proteins, often suggested to be related to neurodegenerative diseases, like amyloid beta A4 protein precursor, superoxide dismutase and apolipoprotein E precursor could only be found in the depleted CSF samples. The effect of a derivatization of tryptic peptides with 2- methoxy-4,5-dihydro-1H-imidazole reagent for protein identification with MS was also employed to increase the number of identified proteins and the sequence coverages. The results presented in this study illustrate the benefit of combining a sample pre-fractionation step and a label's ability to enhance the ionization efficiency with the potential of CE using PolyE-323 modified capillaries in the analysis of complex samples. The straight-forward approach that provides speed and simplicity resulting in high-resolution separations and low sample consumption represents an easily applicable separation technique that can serve as a complement to other currently existing analytical approaches needed in modern proteomic analysis of clinically relevant samples.

Place, publisher, year, edition, pages
2008. Vol. 14, no 4, 249-260 p.
Keyword [en]
CE, MALDI-MS/MS, CSF, proteomics, peptides, immunoaffinity depletion, chemical modification
National Category
Other Basic Medicine
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-98067DOI: 10.1255/ejms.929ISI: 000259337500005PubMedID: 18756023OAI: oai:DiVA.org:uu-98067DiVA: diva2:173237
Available from: 2009-02-20 Created: 2009-02-06 Last updated: 2011-05-13Bibliographically approved
In thesis
1. Surface Modified Capillaries in Capillary Electrophoresis Coupled to Mass Spectrometry: Method Development and Exploration of the Potential of Capillary Electrophoresis as a Proteomic Tool
Open this publication in new window or tab >>Surface Modified Capillaries in Capillary Electrophoresis Coupled to Mass Spectrometry: Method Development and Exploration of the Potential of Capillary Electrophoresis as a Proteomic Tool
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The increased knowledge about the complexity of the physiological processes increases the demand on the analytical techniques employed to explore them. A comprehensive analysis of the entire sample content is today the most common approach to investigate the molecular interplay behind a physiological deviation. For this purpose a method that offers a number of important properties, such as speed and simplicity, high resolution and sensitivity, minimal sample volume requirements, cost efficiency and robustness, possibility of automation, high-throughput and wide application range of analysis is requested. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has a great potential and fulfils many of these criteria. However, further developments and improvements of these techniques and their combination are required to meet the challenges of complex biological samples.

Protein analysis using CE is a challenging task due to protein adsorption to the negatively charged fused-silica capillary wall. This is especially emphasised with increased basicity and size of proteins and peptides. In this thesis, the adsorption problem was addressed by using an in-house developed physically adsorbed polyamine coating, named PolyE-323. The coating procedure is fast and simple that generates a coating stable over a wide pH range, 2-11. By coupling PolyE-323 modified capillaries to MS, either using electrospray ionisation (ESI) or matrix-assisted laser desorption/ionisation (MALDI), successful analysis of peptides, proteins and complex samples, such as protein digests and crude human body fluids were obtained. The possibilities of using CE-MALDI-MS/MS as a proteomic tool, combined with a proper sample preparation, are further demonstrated by applying high-abundant protein depletion in combination with a peptide derivatisation step or isoelectric focusing (IEF). These approaches were applied in profiling of the proteomes of human cerebrospinal fluid (CSF) and human follicular fluid (hFF), respectively. Finally, a multiplexed quantitative proteomic analysis was performed on a set of ventricular cerebrospinal fluid (vCSF) samples from a patient with traumatic brain injury (TBI) to follow relative changes in protein patterns during the recovery process.

The results presented in this thesis confirm the potential of CE, in combination with MS, as a valuable choice in the analysis of complex biological samples and clinical applications.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 603
Keyword
Topology optimization, Design optimization, Material distribution, Wave propagation problems, Inverse problems, Acoustic devices, Medical microwave tomography, High performance computing
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-9554 (URN)978-91-554-7411-9 (ISBN)
Public defence
2009-03-20, B41, BMC, Husargatan 3, Uppsala, 10:15 (English)
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Supervisors
Available from: 2009-02-20 Created: 2009-02-06 Last updated: 2009-03-25Bibliographically approved

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Bergquist, JonasWetterhall, Magnus

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