Internal Energy Distribution in Electrospray Ionization from the Multiple-Collision Model: The case of a Thermal-Like Distribution

In the presented study, the ion survival yields of the theoretical mass spectra simulated by the MassKinetic software are fitted with the experimental ion survival yield of substituted benzylpyridinium cations reported in a precedent paper (J. Mass Spectrom. 1999, 34, 1373-1379). A partially elastic multiple collision model is considered for describing the ion behaviour into the desolation area of the ESI source. The adjusting parameters are not the shape and the position of the P(Eint) curve but rather parameters related to the source acting, such as the pressure and the kinetic energy of ions entering the desolvation zone. In the case of a PE SCIEX ESI source interfaced with a quadrupole mass spectrometer, the energy uptake can then be well-defined by considering the case of a thermal-like distribution an average number of “effective” collisions of 29. From this model, it’s possible to correlate the voltage values applied on the orifice of the desolvation area to initial kinetic energy of ions entering into the collision zone of the ESI source. In the present case, these theoretical initial kinetic energy values range from 5.5 to 9 eV and the results of calculations shown also that the mean internal energy increases linearly with the orifice voltage. This modelling allows defining the internal energy distribution of ions in different part of the ESI source. The activation conditions occurring into the studied ESI source can be compared to a warm-up of ions. Indeed, the internal energy distributions resemble to thermal distribution of ions having a “characteristic temperature” (Tchar) parameter between 1020 to 1550 K. In addition, this study evidences a linear correlation between and Tchar. The slope value of this curve can be related to a calorimetric parameter such as the heat capacity of the activated substituted benzytlpyridinium cations

Model mass spectrometric study of competitive interactions of antimicrobial bisquaternary ammonium drugs and aspirin with membrane phospholipids

The aim of the study is to reveal molecular mechanisms of possible activity modulation of antimicrobial bis-quaternary ammonium compounds (BQAC) and aspirin (ASP) through noncovalent competitive complexation under their combined introduction into the model systems with membrane phospholipids. Methods. Binary and triple systems containing either decamethoxinum or ethonium, or thionium and aspirin, as well as dipalmitoyl-phosphatidylcholine (DPPC) have been investigated by electrospray ionization mass spectrometry. Results. Basing on the analysis of associates recorded in the mass spectra, the types of nonocovalent complexes formed in the systems studied were determined and the supposed role of the complexation in the BQAC and ASP activity modulation was discussed. The formation of associates of BQAC dications with ASP anion is considered as one of the possible ways of deactivation of ionic forms of the medications. The formation of stable complexes of BQAC with DPPC and ASP with DPPC in binary systems as well as the complexes distribution in triple-components systems BQAC:ASP:DPPC point to the existence of competition between drugs of these two types for the binding to DPPC. Conclusions. The results obtained point to the competitive complexation in the model molecular systems containing the BQAC, aspirin and membrane phospholipids. The observed phenomenon testifies to the possibility of modulating the activity of bisquaternary antimicrobial agents and aspirin under their combined usage, due to the competition between the drugs for binding to the target membrane phospholipid molecules and also due to the formation of stable noncovalent complexes between BQAC and ASP

Biomolekulák szerkezetének és kölcsönhatásainak vizsgálata informatikai és tömegspektrometriai módszerek együttes alkalmazásával = Structure and interaction of biomolecules studied by a combined application of mass spectrometry and informatics

A kutatás legfontosabb eredménye peptidek, glikopeptidek és fehérjék tömegspektrometriás viselkedésének tapasztalati és elméleti leírása. Ezen eredmények alapján olyan, nemzetközi összehasonlításban is sikeres módszereket és szoftvereket fejlesztettünk ki, mely a szerkezetkutatás lehetőségeit és a tömegspektrometria alkalmazhatóságát jelentősen bővítik. Meghatároztuk az ütközési energia optimális értéke és a molekulatömeg közötti összefüggést; mely elősegíti a kísérletek automatizálását. Sikeresen leírtuk peptidek és glikopeptidek fragmentációjának szabályait, mely a spektrumok automatikus értelmezésének alapjául szolgál. Ezen eredményeink alapján olyan szoftvert dolgoztunk ki, mely lehetővé teszi a glikoziláció mintázatának meghatározását, ill. amely ezt a rendkívül munkaigényes értékelést automatizálja. Alapkutatási eredményeinket felhasználtuk a glikozilációs mintázat meghatározására, valamint kapcsolatot mutattunk ki egyes betegségek és fehérjék glikozilációja között. | The most significant result of the research project is experimental and theoretical description of the mass spectrometric behaviour of peptides, glycopeptides and proteins. Based on these results we have introduced successful new methodologies and software tools to improve applicability of mass spectrometry for structure analysis and for proteomics. A correlation between the optimal collision energy and molecular mass has been established; which facilitates the design of automatic operation protocols for mass spectrometry. Fragmentation behaviour of peptides and glycopeptides has been described; which is the basis of automatic spectra evaluation. Based on these results a new algorithm and software have been developed, which allows automatic determination of glycosylation patterns. This can substitute the very time consuming manual interpretation; which has been a major obstacle for studying glycosylation. The results described above have been used to determine glycosylation patterns and to observe correlations between protein glycosylation and various illnesses

Fast screening method for wine headspace compounds using solid-phase microextraction (SPME) and capillary GC technique

Solid-phase microextraction (SPME) coupled to capillary gas chromatography-mass spectrometry (GC-MS) was used for determination of volatile wine components. This combination offers a simple, quick, and sensitive approach suitable for characterization of wine aroma compounds without a complicated sample preparation procedure. Wines are characterized by "aromagrams", a set of identified components with corresponding relative abundances. Reproducibility (RSD errors of relative peak abundances) due to the analytical procedure are ca. 4%; variations among different samples of the same type of wine from the same region are ca. 8%. SPME-GC(-MS) has been shown to yield far larger differences among different wine types (Chardonnay, Muscat Ottonel, and Tramini) and among the same type of wine produced in different regions, showing the utility of the technique in wine analysis

Deciphering Salt and Solvent Effects in the Chromatographic Separation of Heparan Sulfate Disaccharides

Heparan sulfate (HS) belongs to the class of glycosaminoglycans, it is a polysaccharide consisting of repeating disaccharide units of hexuronic acid and N-acetylglucosamine. The saccharide units can be sulfated at various positions and epimerization may also occur along the chain. These modifications influence interactions of the HS chain with effector proteins such as cytokines and chemokines. Determining the ratio of these different structures is important in understanding the mechanisms underlying several diseases. Analysis of intact HS chains is practically impossible by instrumental analytical tools due to their large size (up to 70 kDa). Characterization of the average sulfation pattern is usually performed after enzymatic hydrolysis of the polymeric chain into the constituent disaccharide units. However, HPLC-MS analysis of HS disaccharides poses a challenge from both chromatography and mass spectrometry sides, due to their diverse polarity and unfavorable ionization characteristics. The aim of our work was to systematically investigate the chromatographic effects of solvent composition, salt concentration, and salt type in isocratic HILIC-WAX separations of HS disaccharides building on our previous results [1]. Acetonitrile-water ratio of the solvent highly influenced both the elution characteristics and ionization efficiency. Altering the salt concentration improved elution characteristics and did not cause ion suppression. Based on these results, we developed a salt gradient operating with self-packed HILIC-WAX µHPLC columns coupled to ESI mass spectrometry working in negative ion mode. Using the salt gradient improved sensitivity and repeatability could be achieved, compared to previous methods using the same resin. It was possible to separate and quantify the unsaturated HS disaccharides down to a few femtomoles, using a relatively short, 20-minute long gradient. Application of the described method was demonstrated in case of biological examples. Sulfation patterns of heparan sulfates determined using the present method enabled HS structural characterization from limited sample amount

Degrees of freedom effect on fragmentation in tandem mass spectrometry of singly charged supramolecular aggregates of sodium sulfonates

The characteristic collision energy (CCE) to obtain 50% fragmentation of positively and negatively single charged non-covalent clusters has been measured. CCE was found to increase linearly with the degrees of freedom (DoF) of the precursor ion, analogously to that observed for synthetic polymers. This suggests that fragmentation behavior (e.g. energy randomization) in covalent molecules and clusters are similar. Analysis of the slope of CCE with molecular size (DoF) indicates that activation energy of fragmentation of these clusters (loss of a monomer unit) is similar to that of the lowest energy fragmentation of protonated leucine-enkephalin. Positively and negatively charged aggregates behave similarly, but the slope of the CCE vs DoF plot is steeper for positive ions, suggesting that these are more stable than their negative counterparts

Salt and solvent effects in the microscale chromatographic separation of heparan sulfate disaccharides

The analysis of heparan sulfate disaccharides poses a real challenge both from chromatographic and mass spectrometric point of view. This necessitates the constant improvement of their analytical methodology. In the present study, the chromatographic effects of solvent composition, salt concentration, and salt type were systematically investigated in isocratic HILIC-WAX separations of heparan sulfate disaccharides. The combined use of 75% acetonitrile with ammonium formate had overall benefits regarding intensity, detection limits, and peak shape for all salt concentrations investigated. Results obtained with the isocratic measurements suggested the potential use of a salt gradient method in order to maximize separation efficiency. A 3-step gradient from 14 mM to 65 mM ammonium formate concentration proved to be ideal for separation and quantitation. The LOD of the resulting method was 0.8-1.5 fmol for the individual disaccharides and the LOQ was between 2.5-5 fmol. Outstanding linearity could be observed up to 2 pmol. This novel combination provided sufficient sensitivity for disaccharide analysis, which was demonstrated by the analysis of heparan sulfate samples from porcine and bovine origin

Simple correction improving long-term reproducibility of HPLC-MS

Summary Signal intensities in long series of HPLC-MS experiments often vary, which decrease reproducibility and may cause bias in the results. It was found that the sensitivity of various components change differently; in our case variability is in the order of 20-40%; and it is most likely due to changing conditions in ESI ionization. The most often used intensity correction methods do not take this effect into account. The change in signal intensities (peak areas) can be well described by a polynomial function; we found that a 4th order polynomial is most often suitable. We suggest a simple correction algorithm based on polynomial fitting. When the experiments were inherently well reproducible, this correction improved reproducibility from 12% to 3% (on average for various components). When random errors were larger, this improvement was less significant (15% to 12% in nano-ESI), but nevertheless essential in order to avoid possible bias in the results