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Protein content in aqueous humor from patients with pseudoexfoliation (PEX) investigated by capillary-LC MALDI-TOF/TOF MS
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Ophthalmology.
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.
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2009 (English)In: PROTEOMICS - Clinical Applications, ISSN 1862-8346, E-ISSN 1862-8354, Vol. 3, no 3, 299-306 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2009. Vol. 3, no 3, 299-306 p.
Keyword [en]
Aqueous humor, Capillary liquid chromatography, Human body fluids, MALDI-TOF MS/MS, Pseudoexfoliation syndrome
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97814DOI: 10.1002/prca.200780077ISI: 000264794700002OAI: oai:DiVA.org:uu-97814DiVA: diva2:172893
Available from: 2008-11-20 Created: 2008-11-20 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Development of Liquid-based Separation Techniques using Tailored Surfaces for Analysis of Biological Samples
Open this publication in new window or tab >>Development of Liquid-based Separation Techniques using Tailored Surfaces for Analysis of Biological Samples
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Universitetsbiblioteket, 2008. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 577
Keyword
aqueous humor, biological samples, capillary electrophoresis, coatings, drugs, liquid chromatography, molecular imprinted polymers, peptides, proteins, pseudoexfoliation
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-9399 (URN)978-91-554-7350-1 (ISBN)
Public defence
2008-12-11, B42, BMC, Husargatan 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2008-11-20 Created: 2008-11-20 Last updated: 2012-02-08Bibliographically approved
2. Molecular and epidemiological studies on eyes with pseudoexfoliation syndrome
Open this publication in new window or tab >>Molecular and epidemiological studies on eyes with pseudoexfoliation syndrome
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pseudoexfoliation (PEX) syndrome is an age-related condition characterized by the production and accumulation of extracellular fibrillary material in the anterior segment of the eye. PEX predisposes for several pathological conditions, such as glaucoma and complications during and after cataract surgery. The pathogenesis of PEX is not yet fully understood. It is multifactorial with genetics and ageing as contributing factors.

We aimed to study the proteome in aqueous humor (AH) in PEX in order to increase the knowledge about its pathophysiology. Therefore, we developed sampling techniques and evaluated separation methods necessary for analyzing small sample volumes. Other objectives were to study the lens capsule in eyes with PEX regarding small molecules, and to investigate the association between PEX and cataract surgery in a population-based 30-year follow-up study.

Samples of AH from eyes with PEX and control eyes were collected during cataract surgery. In pooled, and individual samples, various liquid based separation techniques and high resolution mass spectrometry were utilized. For quantitation, various methods for labeling, and label free techniques were applied. Lens capsules were collected from some of the patients, and analysed by imaging mass spectrometry. A cohort of 1,471 elderly individuals underwent a comprehensive ophthalmological examination at baseline. Medical information was obtained by questionnaires, and from medical records. Incident cases of cataract surgery were identified by review of medical records.

In the initial study, several techniques were explored for protein detection, and a number of proteins were identified as differentially expressed. In the individually labelled samples, changes in the proteome were observed. Eyes with PEX contained higher levels of proteins involved in inflammation, oxidative stress, and coagulation, suggesting that these mechanisms are involved in the pathogenesis in PEX. The levels of β/γ-crystallins were significantly increased in PEX, which is a novel finding. In the lens capsules from individuals with PEX, changes in the lipid composition was observed with time-of-flight secondary ion mass spectrometry. These changes remain to be elucidated. By multivariate analysis, lens opacities were the first, and PEX the second most important predictor for cataract surgery, the later accounting for a 2.38-fold increased risk for cataract surgery.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1130
Keyword
Pseduoexfoliation syndrome, PEX, aqueous humor, cataract, cataract surgery, lens capsule, proteomics, mass spectrometry, MS, MALDI TOF MS/MS, quantitative proteomics, iTRAQ, dimethyl labeling, imaging mass spectrometry, IMS, time of flight secondary ion mass spectrometry, TOF-SIMS, crystallin, epidemiology, risk factor
National Category
Ophthalmology
Research subject
Ophtalmology
Identifiers
urn:nbn:se:uu:diva-260714 (URN)978-91-554-9312-7 (ISBN)
Public defence
2015-10-15, Fåhreussalen,ingång C5, Rudbecklaboratoriet, Akademiska sjukhuset, Dag Hammarskjölds väg 20, 751 85 Uppsala, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2015-09-24 Created: 2015-08-24 Last updated: 2015-10-01

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Taube, Amelie BotlingAndersson, MaritBergquist, Jonas

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