A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

Very Low-Pressure CID Experiments: High Energy Transfer and Fragmentation Pattern at the Single Collision Regime.

We have performed CID experiments on a triple quadrupole instrument, lowering the collision gas pressure by 50 times compared to its conventional value. The results show that at very low-collision gas pressure, single collisions dominate the spectra. Indirectly, these results suggest that under conventional conditions, 20-50 collisions may be typical in CID experiments. The results show a marked difference between low- and high-pressure CID spectra, the latter being characterized in terms of 'slow heating' and predominance of consecutive reactions. The results indicate that under single collision conditions, the collisional energy transfer efficiency is very high: nearly 100% of the center of mass kinetic energy is converted to internal energy.

Altered Glycosylation of Human Alpha-1-Acid Glycoprotein as a Biomarker for Malignant Melanoma.

A high-resolution HILIC-MS/MS method was developed to analyze anthranilic acid derivatives of N-glycans released from human serum alpha-1-acid glycoprotein (AGP). The method was applied to samples obtained from 18 patients suffering from high-risk malignant melanoma as well as 19 healthy individuals. It enabled the identification of 102 glycan isomers separating isomers that differ only in sialic acid linkage (α-2,3, α-2,6) or in fucose positions (core, antenna). Comparative assessment of the samples revealed that upregulation of certain fucosylated glycans and downregulation of their nonfucosylated counterparts occurred in cancer patients. An increased ratio of isomers with more α-2,6-linked sialic acids was also observed. Linear discriminant analysis (LDA) combining 10 variables with the highest discriminatory power was employed to categorize the samples based on their glycosylation pattern. The performance of the method was tested by cross-validation, resulting in an overall classification success rate of 96.7%. The approach presented here is significantly superior to serological marker S100B protein in terms of sensitivity and negative predictive power in the population studied. Therefore, it may effectively support the diagnosis of malignant melanoma as a biomarker.

Synthesis, characterization and systematic comparison of FITC-labelled GnRH-I, -II and -III analogues on various tumour cells.

Targeted tumour therapy is the focus of recent cancer research. Gonadotropin-releasing hormone (GnRH) analogues are able to deliver anticancer agents selectively into tumour cells, which highly express GnRH receptors. However, the effectiveness of different analogues as targeting moiety in drug delivery systems is rarely compared, and the investigated types of cancer are also limited. Therefore, we prepared selectively labelled, fluorescent derivatives of GnRH-I, -II and -III analogues, which were successfully used for drug targeting. In this manuscript, we investigated these analogues' solubility, stability and passive membrane permeability and compared their cellular uptake by various cancer cells. We found that these labelled GnRH conjugates provide great detectability, without undesired cytotoxicity and passive membrane permeability. The introduced experiments with these conjugates proved their reliable tracking, quantification and comparison. Cellular uptake efficiency was studied on human breast, colon, pancreas and prostate cancer cells (MCF-7, HT-29, BxPC-3, LNCaP) and on dog kidney cells (Madin-Darby canine kidney). Each of the three conjugates was taken up by GnRH-I receptor-expressing cells, but the different cells preferred different analogues. Furthermore, we demonstrated for the first time the high cell surface expression of GnRH-I receptors and the effective cellular uptake of GnRH analogues on human pharynx tumour (Detroit-562) cells. In summary, our presented results detail that the introduced conjugates could be innovative tools for the examination of the GnRH-based drug delivery systems on various cells and offer novel information about these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

[Mass spectrometry: past and present]. / Tömegspektrometria: múlt és jelen.

Mass spectrometry is a highly sensitive high-throughput instrumental analytical technique. It is used to determine the molecular mass, but also gives information on molecular structure amd is used for quantitation as well. Although it was developed over 100 years ago, it continues to evolve, both with respect to figures of merit (like sensitivity) and with respect to applications in various novel fields of science and technology. Mass spectrometry is capable of studying macromolecules (like proteins and protein complexes), and has very high sensitivity, now compounds at the atto- or zeptomol level can also be studied. Mass spectrometry can be coupled to separation techniques, and can be used to analyze complex mixtures, trace level compounds in biological matrices like active pharmaceutical ingredients or metabolites. In recent years in proteomics research has become a major new direction. In the present review we briefly introduce basic mass spectrometry techniques (ion surces, analyzers), combinations with chromatography (GC/MS, HPLC/MS), CEI MS) and tandem mass spectrometry. We also introduce two novel methods, mass spectrometry "imaging" and "lab-on-a-chip" technology.

Electrospray ionization-tandem mass spectrometric study of fused nitrogen-containing ring systems.

Four fused nitrogen-containing ring systems were investigated by electrospray ionization-tandem mass spectrometry: Pyridazino-indoles, pyridazino-quinolines, a pyrimido-quinoline derivative and pyrimido-cinnolines. Fragmentation patterns of these compounds are discussed and compared. Several characteristic cross-ring fragments were formed mainly on the pyridazine and pyrimidine rings of the ring systems. The connected Cl, NO2 , Me, Ph and more extended heterocyclic substituents influenced the fragmentation.

Development and Dissolution Study of a ß-Galactosidase Containing Drinking Straw.

Today, in addition to many different physicochemical and pharmacological properties of the active ingredients and excipients, the developer of a pharmaceutical formulation must take into account several factors during the formulation process in order for the patient to cooperate to use the formulation accurately. One of the innovative solutions in paediatrics may be the use of medicated drinking straws. For our studies, we successfully prepared lactase-containing, rapid disintegration particles by two techniques commonly used in the pharmaceutical industry. The simulation of the usage of the filled straws was presented from a new perspective for the patient by an in vitro method. The effect of the temperature of the liquid used during the administration of the straw and the effect of the frequency during the application on the dissolution rate were investigated. According to our results, in the case of a straw containing rapidly dissolving particles, the temperature of the used liquid and the mode of administration (frequency) play a significant role in the release rate from the composition.

Association of plasma tryptophan concentration with periaqueductal gray matter functional connectivity in migraine patients.

Altered periaqueductal gray matter (PAG) functional connectivity contributes to brain hyperexcitability in migraine. Although tryptophan modulates neurotransmission in PAG projections through its metabolic pathways, the effect of plasma tryptophan on PAG functional connectivity (PAG-FC) in migraine has not been investigated yet. In this study, using a matched case-control design PAG-FC was measured during a resting-state functional magnetic resonance imaging session in migraine without aura patients (n = 27) and healthy controls (n = 27), and its relationship with plasma tryptophan concentration (TRP) was assessed. In addition, correlations of PAG-FC with age at migraine onset, migraine frequency, trait-anxiety and depressive symptoms were tested and the effect of TRP on these correlations was explored. Our results demonstrated that migraineurs had higher TRP compared to controls. In addition, altered PAG-FC in regions responsible for fear-cascade and pain modulation correlated with TRP only in migraineurs. There was no significant correlation in controls. It suggests increased sensitivity to TRP in migraine patients compared to controls. Trait-anxiety and depressive symptoms correlated with PAG-FC in migraine patients, and these correlations were modulated by TRP in regions responsible for emotional aspects of pain processing, but TRP did not interfere with processes that contribute to migraine attack generation or attack frequency.

Citalopram Neuroendocrine Challenge Shows Altered Tryptophan and Kynurenine Metabolism in Migraine.

Altered tryptophan (TRP) metabolism may have an important role in migraine susceptibility through its main metabolites, serotonin and kynurenine (KYN). Both affect pain processing and stress response by interfering with neural and brain hypersensitivity and by interacting with chemokines and cytokines that control vascular and inflammatory processes. The involvement of these pathways in migraine has been widely studied, but acute citalopram neuroendocrine challenge on TRP metabolism and cytokine profile has not been investigated yet. In our study, females with episodic migraine without aura and healthy controls were studied before and after acute citalopram or placebo in a double-blind setting. At baseline, increased TRP/large neutral amino acid (LNAA) ratio and decreased RANTES chemokine concentration were detected in migraine patients compared to controls. The challenge induced a significant increase in TRP, KYN, and TRP/LNAA in healthy controls, but not in migraine patients. Furthermore, migraine attack frequency negatively correlated with KYN/TRP ratio and positively correlated with the neuroendocrine-challenge-induced KYN concentration increase. Our results support a decreased breakdown of TRP via KYN pathway and a failure to modulate TRP-KYN pathway during citalopram-induced acute stress together with an increased vascular sensitivity in migraine. These mechanisms may provide useful drug targets for future drug development.

Optimization and Development of Albumin-Biopolymer Bioconjugates with Solubility-Improving Properties.

Bioconjugation is an emerging field in the food and pharmaceutical industry. Due to its biocompatibility and high ligand binding capacity, albumin is widely used in modern drug delivery systems. However, the protein is sensitive to environmental stresses; albumin conjugates, on the other hand, have improved functional properties. Biopolymers are gaining interest due to their biodegradability and safety, compared to synthetic polymers. In this study, albumin-biopolymer bioconjugates were prepared by nonenzymatic Maillard reaction at 60 °C and 80% relative humidity. This nonenzymatic conjugation takes place between reducing sugars and available amino groups of a protein in certain conditions. The optimal molar ratio and time for the conjugation were studied by several investigation methods, including circular dichroism and fluorescence spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and determination of available amino groups with ortho-phthaldialdehyde (OPA) assay. All of the measurements provided evidence for the covalent bonding of albumin and biopolymers, resulting in bioconjugates. Based on the results, a higher molar ratio and longer time are necessary to complete the reaction with the available amino groups. However, the optimal parameters are specific to each given biopolymer. The rheological behavior of the conjugates is characteristic of the initial biopolymer, which can be useful in drug development. Moreover, both the physical characteristics of albumin and the solubility-improving capacity were enhanced. Therefore, the potential use of albumin-biopolymer bioconjugates in the pharmaceutical industry could be considered.

Investigating thermal stability based on the structural changes of lactase enzyme by several orthogonal methods.

Thermal stability of lactase (ß-galactosidase) enzyme has been studied by a variety of physico-chemical methods. ß-galactosidase is the main active ingredient of medications for lactose intolerance. It is typically produced industrially by the Aspergillus oryzae filamentous fungus. Lactase was used as a model to help understand thermal stability of enzyme-type biopharmaceuticals. Enzyme activity (hydrolyzation of lactose) of ß-galactosidase was determined after storing the solid enzyme substance at various temperatures. For a better understanding of the relationship between structure and activity changes we determined the mass and size of the molecules with gel electrophoresis and dynamic light scattering and detected aggregation processes. A bottom-up proteomic procedure was used to determine the primary amino acid sequence and to investigate changes in the N-glycosylation pattern of the protein. NMR and CD spectroscopic methods were used to observe changes in higher order structures and to reveal relationships between structural and functional changes.

Exposure to Batrachochytrium dendrobatidis affects chemical defences in two anuran amphibians, Rana dalmatina and Bufo bufo.

BACKGROUND: Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, one of the major causes of worldwide amphibian biodiversity loss. Many amphibians exhibit skin-based chemical defences, which may play an important role against invading pathogens, but whether the synthesis of these chemical compounds is enhanced or suppressed in the presence of pathogens is largely unknown. Here we investigated direct and indirect effects of larval exposure to the globally distributed and highly virulent Bd-GPL strain on skin secreted chemical defences and life history traits during early ontogeny of agile frogs (Rana dalmatina) and common toads (Bufo bufo). RESULTS: Exposure to Bd during the larval stage did not result in enhanced synthesis of the antimicrobial peptide Brevinin-1 Da in R. dalmatina tadpoles or in increased production of bufadienolides in B. bufo tadpoles. However, exposure to Bd during the larval stage had a carry-over effect reaching beyond metamorphosis: both R. dalmatina and B. bufo froglets contained smaller quantities of defensive chemicals than their Bd-naïve conspecifics in the control treatment. Prevalence of Bd and infection intensities were very low in both larvae and metamorphs of R. dalmatina, while in B. bufo we observed high Bd prevalence and infection intensities, especially in metamorphs. At the same time, we did not find a significant effect of Bd-exposure on body mass or development rate in larvae or metamorphs in either species. CONCLUSIONS: The lack of detrimental effect of Bd-exposure on life history traits, even parallel with high infection intensities in the case of B. bufo individuals, is surprising and suggests high tolerance of local populations of these two species against Bd. However, the lowered quantity of defensive chemicals may compromise antimicrobial and antipredatory defences of froglets, which may ultimately contribute to population declines also in the absence of conspicuous mass-mortality events.

Composition optimization and stability testing of a parenteral antifungal solution based on a ternary solvent system.

An intravenous solution is a dosage forms intended for administration into the bloodstream. This route is the most rapid and the most bioavailable method of getting drugs into systemic circulation, and therefore it is also the most liable to cause adverse effects. In order to reduce the possibility of side effects and to ensure adequate clinical dosage of the formulation, the primarily formulated composition should be optimized. It is also important that the composition should retain its therapeutic effectiveness and safety throughout the shelf-life of the product. This paper focuses on the optimization and stability testing of a parenteral solution containing miconazole and ketoconazole solubilized with a ternary solvent system as model drugs. Optimization of the solvent system was performed based on assessing the risk/benefit ratio of the composition and its properties upon dilution. Stability tests were conducted based on the EMEA (European Medicines Agency) "guideline on stability testing: stability testing of existing active substances and related finished products". Experiments show that both the amount of co-solvent and surface active agent of the solvent system could substantially be reduced, while still maintaining adequate solubilizing power. It is also shown that the choice of various containers affects the stability of the compositions. It was concluded that by assessing the risk/benefit ratio of solubilizing power versus toxicity, the concentration of excipients could be considerably decreased while still showing a powerful solubilizing effect. It was also shown that a pharmaceutically acceptable shelf-life could be assigned to the composition, indicating good long-term stability.

Development, validation and application of LC-MS/MS method for quantification of amino acids, kynurenine and serotonin in human plasma.

Altered serotonergic neurotransmission is a key factor in several neurologic and psychiatric disorders such as migraine. Human and animal studies suggest that chronically low interictal serotonin levels of plasma and brain may facilitate increased activity of the trigeminovascular pathway, and may contribute to development of repeated migraine attacks. However, brain serotonin synthesis is affected by the concentration of tryptophan, its metabolites and a number of amino acids. In this work a simple and robust LC-MS/MS method for the quantitative determination of valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serotonin and kynurenine in human plasma has been developed and validated. Sample preparation was achieved by protein precipitation, using trifluoroacetic acid. Chromatographic separation was carried out on a Supelco Ascentis® Express C18 column (3.0 mm i.d. × 150 mm, 2.7 µm) equipped with an Agilent Zorbax Eclipse XDB C8 guard-column under isocratic conditions at a flow rate of 0.4 mL/min, over a 6.5 min run time. Mobile phase was 0.2% trifluoroacetic acid - acetonitrile (85:15, v/v). The eight analytes and two internal standards were ionized by positive electrospray ionization and detected in multiple reaction monitoring mode. A "fit-for-purpose" validation approach was adopted using surrogate matrix for the preparation of calibration samples. The calibration curves of all analytes showed excellent linearities with a correlation coefficient (r2) of 0.998 or better. Spiked surrogate matrix samples and pooled human plasma were used as quality control samples. Intra-day and inter-day precisions were less than 11.8% and 14.3%, and accuracies were within the ranges of 87.4-114.3% and 87.7-113.3%, respectively. Stability of the components in standard solutions, surrogate matrix, pooled plasma and processed samples were found to be acceptable under all relevant conditions. No significant carryover effect was observed. The surrogate matrix behaved parallel to human plasma when assessed by standard addition method and diluting the authentic matrix with surrogate matrix. The method was successfully applied for analysis of 800 human plasma samples to support a clinical study.

Investigation of genetic variants of alpha-1 acid glycoprotein by ultra-performance liquid chromatography-mass spectrometry.

Genetic variants of human plasma alpha-1 acid glycoprotein (AGP) have been studied in cancer, compared with a group of healthy control. AGP has four genetic variants: AGP F1, F2, and S variants correspond to the ORM1 gene whereas AGP A corresponds to the ORM2 gene. The proportion of ORM1 and ORM2 variants were studied in plasma using a novel UPLC-MS method. Plasma total AGP level was 0.5 +/- 0.2 g L(-1) and the proportions of the ORM1 and ORM2 variants were 76.3 +/- 8.2% and 23.7 +/- 8.2%, respectively. In cancer plasma AGP levels increased fourfold and the proportion of ORM1 variants increased to 88.7 +/- 6.8%. Changes in the proportion of genetic variants due to cancer were clearly significant, as shown by statistical analysis. Three different cancer types have been studied, lymphoma, melanoma, and ovarian cancer. The results did not show any difference depending on cancer type. The results indicate that, in accordance with prior expectations, the ORM1 variant is predominantly responsible for the acute-phase property of AGP.

[Difficulties encountered during formulation of a parenteral dosage form containing a poorly soluble drug]. / Rossz vízoldékonyságú hatóanyagok parenterális gyógyszerformaként történo formulálásánál felmerülo problémák.

Poor water solubility and consequently the difficulties in formulating a liquid dosage form is a great concern in pharmaceutical development. The importance of this issue is underlined by the fact that 10-30% of marketed drugs and 60-70% of drugs coming from early development stage have solubility problems. In this paper we summarize the existing solubility enhancing techniques that are applicable in parenteral dosage forms for overcoming the issue. We address the problem of choosing the most adequate solubility enhancing technique and present the considerations that should be kept in mind during formulating the solvent systems. Such questions are for example the possible haemolysing effect of the excipients, pH of the composition and its compatibility with various sterilizing methods. We also focus on the probable technological issues, which may arise in each solubility enhancing method, we present examples for every one of them and where possible the solution to the problem is also proposed.

[In vivo effect of food on absorption of fluoroquinolones]. / Fluorokinolonok in vivo étel-interakciós vizsgálatai.

Although food-drug interactions have been studied extensively in recent years, in the light of the complex nature of these interactions general guideline for clinical practice can not be given. Drug interactions with food (containing multivalent metal ions or protein) can have an influence on drug absorption with widely variety of mechanism, resulting in changes in both the rate and extent of bioavailability. Food-drug interaction can be important in the clinical practice. Studies of the interaction between food/juice and fluoroquinolones have produced conflicting results. A number of studies give evidence that fluoroquinolones forming slightly soluble complex with metal ions of food show reduced bioavailability. In the same time, concurrent ingestion of food/ juice with fluoroquinolones has been shown not to interfere with their absorption to a clinically significant degree.

Scope and limitation of propylene carbonate as a sustainable solvent in the Suzuki-Miyaura reaction.

The Suzuki-Miyaura reaction is one of the most used transformations in drug research. Thus making this reaction more sustainable is of considerable current interest. Here we show that propylene carbonate (PC) can be used as a solvent for the Suzuki-Miyaura reaction. PC is one of the greenest solvents since it is synthesized under green conditions by the use of carbon dioxide in the air. All reactions proceeded well and good or excellent yields were observed for the biaryl products. Nonetheless in the case of pyridazinones, 2-hydroxypropyl- chain containing side-products were observed. Importantly, this fact allowed the isolation of several novel compounds which were generated under prominently green conditions.

Analysis of complex oligosaccharides using graphitized carbon liquid chromatography/mass spectrometry.

The sugar fraction of alpha-1 acid glycoprotein (AGP) was studied using porous graphitized carbon (PGC) chromatography coupled to mass spectrometry. Electrospray ionization provides efficient control over fragmentation; at low collision energy only molecular species were observed, allowing accurate oligosaccharide profiling. PGC chromatography was useful separating 18 sugars differing in monosaccharide composition. Most of these were separated into several isomeric forms; altogether 49 different oligosaccharides were found in AGP.