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CE MALDI-TOF/TOF MS for multiplexed quantification of proteins in human ventricular cerebrospinal fluid
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, 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, Analytical Chemistry.
2009 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 30, no 10, 1836-1843 p.Article in journal (Refereed) Published
Abstract [en]

CE, interfaced off-line to MALDI-TOF/TOF MS, was for the first time used to quantitatively monitor the protein content in complex   biological and clinical samples with iTRAQ (TM) labeling. The   usefulness and advantage of iTRAQ (TM) labeling, in combination, with   CE MALDI-TOF/TOF MS is demonstrated on mixtures of protein standards and by a case study on human ventricular cerebrospinal fluid samples collected from a patient with traumatic brain injury during patient recovery. Mixtures of five standard proteins were initially analyzed to optimize the experimental conditions for the CE MALDI-MS and MS/MS  analysis. The interactions of proteins and peptides with the capillary   inner wall during CE separation were minimized using PolyE-323 modified  capillaries. The analysis of the ventricular cerebrospinal fluid   samples yielded 43 significantly (p < 0.05 MudPIT scoring) identified   proteins that could be quantitatively monitored over time. The identified changes in protein levels for several of these proteins are well in line with the reports from previous studies on protein patterns that could be related to the post-traumatic processes of traumatic brain injury. This study shows that the presented approach, combining  isobaric tags with CE MALDI-TOF/TOF MS, is a useful choice for quantitative proteomic analysis.

Place, publisher, year, edition, pages
2009. Vol. 30, no 10, 1836-1843 p.
Keyword [en]
CE MALDI-TOF/TOF MS, iTRAQ (TM), Multiplexed quantification, Proteomics, Ventricular cerebrospinal fluid
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-98069DOI: 10.1002/elps.200800714ISI: 000266524500026PubMedID: 19441030OAI: oai:DiVA.org:uu-98069DiVA: diva2:173239
Available from: 2009-02-20 Created: 2009-02-06 Last updated: 2017-12-13Bibliographically 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)
Opponent
Supervisors
Available from: 2009-02-20 Created: 2009-02-06 Last updated: 2009-03-25Bibliographically approved

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Wetterhall, MagnusBergquist, Jonas

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