László Drahos

Comparison of the Internal Energy Distributions of Ions Produced by Different Electrospray Sources

The internal energies of the emitted ions can be modulated in an electrospray source through different experimental conditions. However, the fragmentation pattern depends also on conditions that cannot be controlled by the operator, e.g. the geometry of the source or the mode of transfer of the ions. These differences make difficult the comparison of the electrospray mass spectra obtained with different mass spectrometers. A method for the calibration of the internal energies of ions produced by an electrospray source has been presented previously to study the influence of experimental conditions on the internal energies of the ions. The method permits the calibration of individual working conditions. In this work, new results were obtained with modified values of fragmentation energy taking into account the kinetic shift of the thermometer ions. The internal energy distributions are compared for different instruments with different geometries, and are measured under different accelerating and focusing conditions.

How closely related are the effective and the real temperature

In the context of the kinetic method, the effective temperature is defined as being equal to the real temperature of a population of cluster ions in thermal equilibrium, which would yield the same product ratio as that observed in the mass spectrometric experiment. The effective temperature is not a thermodynamic quantity and depends not only on the internal energy content but also on the experimental conditions. A very good correlation has been found between the effective temperature and the mean internal energy of ions dissociating in the time window of analysis, but not with the mean internal (or excess) energy of the whole ion population. Calculations suggest that assuming a high internal energy is unnecessary for the kinetic method to be accurate. Care should be taken, however, in evalulating entropy effects and to check that the cluster ions studied have the same (or a similar) effective temperature. Copyright

Applications of liquid chromatography–mass spectrometry for food analysis

HPLC-MS applications in the agrifood sector are among the fastest developing fields in science and industry. The present tutorial mini-review briefly describes this analytical methodology: HPLC, UHPLC, nano-HPLC on one hand, mass spectrometry (MS) and tandem mass spectrometry (MS/MS) on the other hand. Analytical results are grouped together based on the type of chemicals analyzed (lipids, carbohydrates, glycoproteins, vitamins, flavonoids, mycotoxins, pesticides, allergens and food additives). Results are also shown for various types of food (ham, cheese, milk, cereals, olive oil and wines). Although it is not an exhaustive list, it illustrates the main current directions of applications. Finally, one of the most important features, the characterization of food quality (including problems of authentication and adulteration) is discussed, together with a future outlook on future directions.

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.

Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection

Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.

Size effect on fragmentation in tandem mass spectrometry

The collision energy or collision voltage necessary to obtain 50% fragmentation (characteristic collision energy/voltage, CCE or CCV) has been systematically determined for different types of molecules [poly(ethylene glycols) (PEG), poly(tetrahydrofuran) (PTHF), and peptides] over a wide mass (degrees of freedom) range. In the case of lithium-cationized PEGs a clear linear correlation (R(2) > 0.996) has been found between CCE and precursor ion mass on various instrument types up to 4.5 kDa. A similar linear correlation was observed between CCV and the mass-to-charge ratio. For singly and multiply charged polymers studied under a variety of experimental conditions and on several instruments, all data were plotted together and showed correlation coefficient R(2) = 0.991. A prerequisite to observe such a good linear correlation is that the energy and entropy of activation in a class of polymers is likely to remain constant. When compounds of different structure are compared, the CCV will depend not only on the molecular mass but the activation energy and entropy as well. This finding has both theoretical and practical importance. From a theoretical point of view it suggests fast energy randomization up to at least 4.5 kDa so that statistical rate theories are applicable in this range. These results also suggest an easy method for instrument tuning for high-throughput structural characterization through tandem MS: after a standard compound is measured, the optimum excitation voltage is in a simple proportion with the mass of any structurally similar analyte at constant experimental conditions.

GlycoMiner: a new software tool to elucidate glycopeptide composition

New computer software, GlycoMiner, has been developed to automatically identify tandem (MS/MS) spectra obtained in liquid chromatography/mass spectrometry (LC/MS) runs which correspond to N-glycopeptides. The program complements conventional proteomics analysis, and can be used in a high-throughput environment. The program interprets the spectra and determines the structure of the corresponding glycopeptides. GlycoMiner runs under Windows, can process spectra obtained on various instruments, and can be downloaded from our website (w3.chemres.hu/ms/glycominer). The algorithm works similarly to a human expert; evaluates the low mass oxonium ions; deduces oligosaccharide losses from the protonated molecule; and identifies the mass of the peptide residue. The program has been tested on tryptic digests of two glycopeptides: AGP (which has five different N-glycosylation sites) and transferrin (with two N-glycosylation sites). Results have been evaluated both manually and by GlycoMiner. Out of 3132 MS/MS spectra 338 were found to correspond to glycopeptides; identification by GlycoMiner showed a 0.1% false positive and 0.1% false negative rate. From these it was possible to identify 196 glycan structures manually; GlycoMiner correctly identified all of these, with no false positives. The rest were low quality spectra, not suitable for structure assignment.

Mass spectrometric and linear discriminant analysis of N-glycans of human serum alpha-1-acid glycoprotein in cancer patients and healthy individuals

N-glycan oligosaccharides of human serum alpha(1)-acid glycoprotein (AGP) samples isolated from 43 individuals (healthy individuals and patients with lymphoma and with ovarian tumor) were analyzed by MALDI-TOF mass spectrometry and a multivariate statistical method (linear discriminant analysis, LDA). 34 different glycan structures have been identified. From the glycosylation pattern determined by mass spectrometry fucosylation and branching indices have been calculated. These parameters show only small differences between the patient groups studied, but these differences are not sufficiently large to use as a potential biomarker. LDA analysis, on the other hand shows a very good separation between the three groups (with a classification of 88%). Cross-validation indicates that the method has predictive power: Identifying cancerous vs. healthy individuals shows 96% selectivity and 93% specificity; identification of lymphoma vs. the mixed group of healthy and ovarian tumor cases is also promising (72% selectivity and 84% specificity). The pilot study presented here demonstrates that mass spectrometry combined with linear discriminant analysis (LDA) may provide valuable data for identifying and studying the pathophysiology of malignant diseases.

Double Diastereocontrol in Bifunctional Thiourea Organocatalysis: Iterative Michael–Michael–Henry Sequence Regulated by the Configuration of Chiral Catalysts

The importance and reactivity consequences of the double diastereocontrol in noncovalent bifunctional organocatalysis were studied. The results suggest that the bifunctional thioureas can have synthetic limitations in multicomponent domino or autotandem catalysis. Nevertheless, we provided a means to exploit this behavior and used the configuration of the chiral catalyst as a control element in organo-sequential reactions.

Novel Synthesis of Phosphinates by the Microwave-Assisted Esterification of Phosphinic Acids

Abstract 1-Hydroxy-3-phospholene oxides (1 and 3) and phenyl-H-phosphinic acid (6) are converted to the corresponding phosphinic esters (2, 4, and 7, respectively) by reaction with simple alcohols on microwave irradiation. Under traditional heating conditions, the esterification does not take place, as in the cases of 1 and 3, or is highly incomplete, as in the case of 6. Steric hindrance in diphenylphosphinic acid prevents efficient microwave-assisted esterification.