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Surface modified capillary electrophoresis combined with in solution isoelectric focusing and MALDI-TOF/TOF MS: A gel-free multidimensional electrophoresis approach for proteomic profiling — Exemplified on human follicular 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.
2009 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1216, no 17, 3621-3628 p.Article in journal (Refereed) Published
Abstract [en]

Development of miniaturized analytical tools continues to be of great interest to face the challenges in proteomic analysis of complex biological samples such as human body fluids. In the light of these challenges, special emphasis is put on the speed and simplicity of newly designed technological approaches as well as the need for cost efficiency and low sample consumption. In this study, we present an alternative multidimensional bottom-up approach for proteomic profiling for fast, efficient and sensitive protein analysis in complex biological matrices. The presented setup was based on sample pre-fractionation using microscale in solution isoelectric focusing (IEF) followed by tryptic digestion and subsequent capillary electrophoresis (CE) coupled off-line to matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS). For high performance CE-separation, PolyE-323 modified capillaries were applied to minimize analyte-wall interactions. The potential of the analytical setup was demonstrated on human follicular fluid (hFF) representing a typical complex human body fluid with clinical implication. The obtained results show significant identification of 73 unique proteins (identified at 95% significance level), including mostly acute phase proteins but also protein identities that are well known to be extensively involved in follicular development.

Place, publisher, year, edition, pages
2009. Vol. 1216, no 17, 3621-3628 p.
Keyword [en]
CE, Human follicular fluid (hFF), MALDI TOF MS/MS, Proteomics, Solution phase IEF
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-98068DOI: 10.1016/j.chroma.2008.12.026ISI: 000265467200005PubMedID: 19155017OAI: oai:DiVA.org:uu-98068DiVA: diva2:173238
Available from: 2009-02-20 Created: 2009-02-06 Last updated: 2011-03-30Bibliographically 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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