Proteomic analysis of the aqueous humour in eyes with pseudoexfoliation syndrome

Background/aimsPseudoexfoliation syndrome (PEX) is characterised by the production and accumulation of extracellular fibrillar material in the anterior segment of the eye. The pathogenesis of PEX is multifactorial with genetic factors and ageing as contributing factors. Previously, an increased concentration of beta-crystalline B2 (CRYBB2) was observed in the aqueous humour (AH) in eyes with PEX in a pooled material. Here, the protein content was examined on individual basis.MethodsDuring cataract surgery, AH was sampled from patients with and without PEX, 10 eyes in each group. The proteins were digested and labelled with isotopomeric dimethyl labels, separated with high-pressure liquid chromatography and analysed in an Orbitrap mass analyzer.ResultsThe concentration of complement factor 3, kininogen-1, antithrombin III and vitamin D-binding protein was increased in all eyes with PEX. Retinol-binding protein 3, glutathione peroxidase, calsyntenin-1 and carboxypeptidase E were decreased in eyes with PEX. Beta-crystalline B1 and CRYBB2 and gamma-crystalline D were up to eightfold upregulated in 4 of 10 in eyes with PEX.Conclusion The results indicate that oxidative stress and inflammation are contributing factors in the formation of PEX. Knowledge about the proteome in PEX is relevant for understanding this condition.

Coupling Ion Chromatography to Q-Orbitrap for the Fast and Robust Analysis of Anionic Pesticides in Fruits and Vegetables

Ion chromatography coupled to a quadrupole Orbitrap mass analyzer was used to develop a multiresidue method for the determination of highly polar pesticides and their metabolites (chlorate, perchlorate, fosetyl-aluminum, glyphosate, aminomethylphosphonic acid (AMPA), phosphonic acid, N-acetyl AMPA, and N-acetyl glyphosate) in fruits and vegetables. After extraction with methanol, samples were diluted 5× with water. No derivatization was applied. Pesticides were separated in an anion-exchange column. Water was used as the ion chromatography mobile phase. A gradient was created by increasing the concentration of KOH in the mobile phase. Ion chromatography provided good and stable retention and separation for all studied compounds. All investigated pesticides had an LOQ of 0.01 mg/kg and a linear range of 0.01–0.50 mg/kg. The ion ratio of the m/z ions produced was stable and adequate (deviation <30%) in all cases. The obtained mass errors (always in full-scan MS and MS2 mode) were <0.2 mDa. The high resolution (>100 000) provided by the Orbitrap analyzer with the low m/z ions obtained (e.g., m/z 80) was effective in obtaining low background matrix signals. The influence of postcolumn infusion of organic solvent on sensitivity was investigated. Acetonitrile was found to be more effective than methanol, increasing the sensitivity 3× with respect to water. The method was validated for five vegetable-based matrixes. Both the sample processing and the analytical measurement were very fast. Hence, the methodology is ideal for high-throughput work.

Coupling liquid chromatography to Orbitrap mass spectrometry

The Orbitrap mass analyzer has become a mainstream mass spectrometry technique. In addition to providinga brief introduction to the Orbitrap technology and its continuing development, this article reviewsthe most recent publications quoting the use of the Orbitrap detection for a variety of chromatographicseparation techniques. Its coupling to reversed-phase liquid chromatography (LC) represents undoubtedlythe most ubiquitous approach to both small molecule and proteomic analyses. Multi-dimensional LCseparations have an important role to play in the proteomics applications while an ultra-high-pressureLC is more frequently encountered in the area of metabolomics and metabolite analysis. Recently, specialchromatographic techniques such as hydrophilic interaction chromatography and its variations have alsobeen also cited with the Orbitrap detection.

Isotope ratio characteristics and sensitivity for uranium determinations using a liquid sampling-atmospheric pressure glow discharge ion source coupled to an Orbitrap mass analyzer

The coupling of an LS-APGD microplasma with an Orbitrap analyzer is applied to the isotope ratio analysis of uranium.