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- Hardenborg, Emilia, 1974-
(författare)
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Development of Liquid-based Separation Techniques using Tailored Surfaces for Analysis of Biological Samples
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Development and improvement of analytical techniques are vital in analytical chemistry research. This thesis describes the development and use of tailored surfaces for bioanalytical applications. In sample preparation, solid phase extraction is often used and the development of a protocol for extraction on a molecular imprinted polymer (MISPE) directly from plasma sample is presented. Molecular imprinted polymers (MIP) offer selective sorbents for the imprinted analyte. MISPE has mainly been used in organic phase but in this thesis the development of a protocol for direct extraction of the analyte form an aqueous phase is described. For analysis of complex samples a separation step is often needed. The growing interest in analysis of biological samples and analysis of the human proteome and potential biomarkers has increased the interest in developing new separation techniques. Capillary electrophoresis (CE) has evolved into an important technique for use in analysis of body fluids. In this thesis a novel polyamine coating named PolyE 323 tailored for minimizing the adsorption of basic proteins to the surface is introduced. A straightforward coating protocol, with four simple rinsing steps, was developed. The coating was highly reproducible and useable over a wide pH range. Successful protein separations on PolyE-323-coated capillaries coupled to electrospray ionization mass spectrometry (ESI-MS) were demonstrated. The coated capillaries were also used in studies of protein content of aqueous humor samples from cataract patients as a complement to capillary liquid chromatography. In the studies presented the protein content of aqueous humor samples from two clinical groups was compared. By using capillary liquid separation techniques coupled to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and MS/MS in combination with isobaric tags for relative and absolute quantitation (iTRAQ) the identity and relative concentrations of proteins in the samples were evaluated. Earlier studies of the proteins in these kinds of samples have mainly been done with techniques using immunological detection where the proteins of interest were chosen in advance. In this thesis it was shown that liquid-based separation techniques are a good complement and by using the mass spectrometry approach presented the protein content of the samples could be evaluated without bias.
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- Hardenborg, Emilia, et al.
(författare)
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Protein content in aqueous humor from patients with pseudoexfoliation (PEX) investigated by capillary-LC MALDI-TOF/TOF MS
- 2009
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Ingår i: PROTEOMICS - Clinical Applications. - : Wiley. - 1862-8346 .- 1862-8354. ; 3:3, s. 299-306
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of proteins in human body fluids is challenging since the composition of the sample often is rather complex. Here we present a method for analysis of proteins in aqueous humor from two groups of cataract patients, with and without pseudoexfoliation (PEX). Aqueous humor is an extracellular fluid contained in the anterior chamber of the eye between the cornea and iris. The limited volume of sample requires sophisticated analysis techniques. Our method is based on a total tryptic digestion of the sample followed by capillary LC-MALDI MS and MS/MS analysis of the peptides. The method is rapid, efficient and suitable as a complement or alternative to more commonly used methods based on gel electrophoretic experiments. With this method we found and unambiguously identified 30 nonredundant proteins. Proteins found include general transport proteins such as albumin and apolipoprotein A1 but also specific proteins involved in immune response, such as complement factors. Cystatin C, clusterin, and crystallins were also found. Although the number of proteins was roughly the same in both groups there was a significant difference in their identities. These findings may give some new insights into the pathophysiology of the PEX syndrome.
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- Taube, Amelie Botling, et al.
(författare)
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Proteins in aqueous humor from cataract patients with and without pseudoexfoliation syndrome.
- 2012
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Ingår i: European Journal of Mass Spectrometry. - : SAGE Publications. - 1469-0667 .- 1751-6838. ; 18:6, s. 531-41
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to investigate the protein content in aqueous humor in eyes with and without pseudoexfoliations (PEX) and to evaluate the quantitative proteomics method, isobaric tagging for relative and absolute protein quantification (iTRAQ), in combination with two separation methods followed by matrix-assisted Laser desorption/ionization (MALDI) mass spectrometry and tandem mass spectrometry (MS/MS). During cataract surgery, samples of aqueous humor were collected from 20 eyes with PEX and from 18 control eyes. The relative concentrations of proteins in the pooled samples of ten PEX eyes and eight controls were evaluated after trypsin digestion and Labeling of the peptides with (iTRAQ) reagent. Two separation methods, Liquid chromatography (LC) and capillary electrophoresis (CE) were used, followed by MALDI mass spectrometry and MS/MS. Furthermore, 1D gel electrophoresis was performed on the remaining ten pooled PEX samples and ten control samples. The gel material was separated by nano-liquid chromatography (nano-LC) followed by Linear-ion-trap quadrupole Fourier transformation ion cyclotron resonance (FT-ICR). Fifty four proteins were identified in the LC runs and 24 with CE. The relative concentrations of beta-crystallines B2 and S were raised and those of angiotensinogen and osteopontin Lowered in the PEX sample compared to the control. The trends regarding beta-crystallines B2, angiotensinogen and osteopontin were confirmed by the 1D gel electrophoresis.
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- Ullsten, Sara, et al.
(författare)
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A polyamine coating for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry of proteins and peptides.
- 2004
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 25:13, s. 2090-2099
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Tidskriftsartikel (refereegranskat)abstract
- A procedure for enhanced capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) of proteins is presented. The use of a newly presented capillary coating, PolyE-323, provided fast separations of typically a few minutes with high efficiency, good deactivation, and no bleeding into the mass spectrometer. Capillaries coated with PolyE-323 showed high stability over a range of pH 2-10, and tolerance towards methanol and acetonitrile, two modifiers commonly used in CE-ESI-MS. Due to the speed and simplicity of the coating procedure, the polymeric surface could, if necessary, easily be regenerated. This capability is especially valuable when working with samples of complex matrix, where a capillary surface cleaning step might be desired in order to eliminate possible memory effects. The potential of PolyE-323-coated capillaries in bioanalysis using CE-ESI-MS was demonstrated by analyzing peptides and proteins up to 66 kDa using time of flight (TOF)-MS. Due to the stable, anodal electroosmotic flow generated by the coating, the use of a sheathless ESI interface was enabled, demonstrated in peptide analysis with attomole sensitivity. The fast on-line CE-ESI-TOF system using PolyE-323-coated capillaries provided efficient separation and detection of a large number of peaks in a short time, exemplified by the analysis of a tryptic digest of bovine serum albumin (BSA). The capability of the developed capillary surface coating was demonstrated by the separation of human plasma and cerebrospinal fluid (CSF).
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