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Proteins in aqueous humor from cataract patients with and without pseudoexfoliation syndrome
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 Chemistry - BMC, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
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2012 (English)In: European journal of mass spectrometry, ISSN 1469-0667, Vol. 18, no 6, 531-541 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2012. Vol. 18, no 6, 531-541 p.
Keyword [en]
pseudoexfoliations (PEX), isobaric tagging, protein quantification, proteomics, aqueous humour, osteopontin, angiotensinogen
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-197670DOI: 10.1255/ejms.1208ISI: 000315745600007OAI: oai:DiVA.org:uu-197670DiVA: diva2:613819
Available from: 2013-04-02 Created: 2013-04-02 Last updated: 2015-10-01Bibliographically approved
In thesis
1. 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1130
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
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Research subject
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)
Available from: 2015-09-24 Created: 2015-08-24 Last updated: 2015-10-01

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