Károly Vékey

Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle) signatures in joint diseases.

Introduction Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. Methods In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. Results EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3+ and CD8+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (pu200a=u200a0.027 and pu200a=u200a0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (pu200a=u200a0.009, after Bonferroni correction). Conclusions Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

Proteomic characterization of thymocyte-derived microvesicles and apoptotic bodies in BALB/c mice

Several studies have characterized exosomes derived from different cell sources. In this work we set the goal of proteomic characterization of two less studied populations of membrane vesicles, microvesicles (100-800 nm) and apoptotic bodies (> 800 nm) released by thymus cells of BALB/c mice. The vesicles were isolated by the combination of differential centrifugation and gravity driven multistep filtration of the supernatant of thymus cell cultures. The size distribution of vesicle preparations was determined by transmission electron microscopy. Proteins were released from the vesicles, digested in solution, and analyzed using nano-HPLC/MS(MS). Ingenuity pathway analysis was used to identify functions related to membrane vesicle proteins. In apoptotic bodies and microvesicles we have identified 142 and 195 proteins, respectively. A striking overlap was detected between the proteomic compositions of the two subcellular structures as 108 proteins were detected in both preparations. Identified proteins included autoantigens implicated in human autoimmune diseases, key regulators of T-cell activation, molecules involved in known immune functions or in leukocyte rolling and transendothelial transmigration. The presence and abundance of proteins with high immunological relevance within thymocyte-derived apoptotic bodies and microvesicles raise the possibility that these subcellular structures may substantially modulate T-cell maturation processes within the thymus.

Hydrogen/deuterium exchange of electrosprayed ions in the atmospheric interface of a commercial triple–quadrupole mass spectrometer

Abstract A novel H/D exchange technique capable of the deuteration of electrosprayed ions has been developed. H/D exchange was carried out by introducing deuterating agent (e.g., d-MeOH) into the curtain gas flow of a commercial triple quadrupole mass spectrometer. In contrast to the widely used H/D exchange techniques, the ions are not trapped in this case. The main advantages of this technique are the ease of use and applicability on most commercial mass spectrometers, including quadrupole-type instruments. Effect of various instrumental parameters was investigated in detail, including spray voltage, spray position, partial pressure of the deuterating agent in the curtain gas, gas flow rates, the orifice-to-skimmer potential and the source temperature. Among these only the partial pressure of the deuterating agent in curtain gas, orifice-to-skimmer potential and source temperature influenced the efficiency of H/D exchange. These suggest that the H/D exchange is likely to occur in the fore-vacuum region of the atmospheric interface. Analytical capabilities of the technique were demonstrated by differentiation of lysine and glutamine protonated molecular ions. Selective quantitation of lysine and glutamine mixtures was achieved, with a lower limit of detection of 1.5% for glutamine and 0.2% for lysine. H/D exchange of multiply charged macromolecular ions can also be carried out using this technique, which was demonstrated using cytochrome c .

Determination of the cultivar and aging of Sicilian olive oils using HPLC-MS and linear discriminant analysis

A large number of certified samples (84) of Sicilian olive oils arising from the eight cultivars most represented in Sicily (Biancolilla, Cerasuola, Moresca, Nocellara del Belice, Nocellara Etnea, Oglialora Messinese, Brandofino and Tonda Iblea) have been collected and analyzed by HPLC/MS using an atmospheric pressure chemical ionization (APCI) source. The sample preparation is very simple; in fact, the oil samples are diluted without any chemical derivatization. A following statistical data treatment by general discriminant analysis (GDA) allows the determination of the olive oil cultivar. Furthermore, changes in the composition of glyceridic components of the olive oils lead to easy discrimination between fresh oils and 1-year-old samples.