Tandem mass spectrometry of isomeric aniline-labeled N-glycans separated on porous graphitic carbon: Revealing the attachment position of terminal sialic acids and structures of neutral glycans.

RATIONALE: Quantitative monitoring of changes in the N-glycome upon disease has gained significance in the context of biomarker discovery. Separation and quantification of isobaric glycan isomers can be attained by using high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS). Collision-induced dissociation (CID)-based fragmentation of separated isobaric glycans is evaluated in respect to its potential of providing fragment ions specific for the linkage positions of terminal sialic acids and the presence of intersecting GlcNAc moieties, respectively. METHODS: N-Glycans were labeled via reductive amination using (12)C6-aniline and (13)C6-aniline as isotope-coded labeling reagents. The differently labeled glycans were merged and separated into various species using a porous graphitic carbon (PGC) stationary phase. Identification of structural features of separated isobaric isomers was performed by CID-based tandem mass spectrometry (MS/MS) carried out in a quadrupole time-of-flight (QqTOF) or a quadrupole ion-trap (IT) mass spectrometer. RESULTS: Working in the negative ion mode, new diagnostic CID fragment ions could be found that are indicative for the α2,6-type linkage of sialic acids. Other diagnostic ions, identified before as being indicative for the substitution of the 6-antenna, could be confirmed as being of relevance also in the case of aniline labeling. In the positive ion mode, CID fragment ions indicative for the structure of short neutral N-glycans were identified. CONCLUSIONS: One new diagnostic ion specific for the linkage position of the terminal sialic acids and one for the presence of bisecting GlcNAc in N-glycans were identified. The aniline label introduced for improved relative quantitation in MS(1) was found not to significantly alter the CID fragmentation patterns that were reported previously by other authors for unlabeled/reduced glycans or for glycans with more polar labels.

Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography-electrospray ionization-mass spectrometry with graphitic carbon stationary phase.

Glycan reductive isotope labeling (GRIL) using (12)C6-/(13)C6-aniline as labeling reagent is reported with the aim of quantitative N-glycan fingerprinting. Porous graphitized carbon (PGC) as stationary phase in capillary scale HPLC coupled to electrospray mass spectrometry with time of flight analyzer was applied for the determination of labeled N-glycans released from glycoproteins. The main benefit of using stable isotope-coding in the context of comparative glycomics lies in the improved accuracy and precision of the quantitative analysis in combined samples and in the potential of correcting for structure-dependent incomplete enzymatic release of oligosaccharides when comparing identical target proteins. The method was validated with respect to mobile phase parameters, reproducibility, accuracy, linearity and limit of detection/quantification (LOD/LOQ) using test glycoproteins. It is shown that the developed method is capable of determining relative amounts of N-glycans (including isomers) comparing two samples in one single HPLC-MS run. The analytical potential and usefulness of GRIL in combination with PGC-ESI-TOF-MS is demonstrated comparing glycosylation in human monoclonal antibodies produced in Chinese hamster ovary cells (CHO) and hybridoma cell lines.

Cloning and functional expression in E. coli of a polyphenol oxidase transcript from Coreopsis grandiflora involved in aurone formation.

Polyphenol oxidases are involved in aurone biosynthesis but the gene responsible for 4-deoxyaurone formation in Asteraceae was so far unknown. Three novel full-length cDNA sequences were isolated from Coreopsis grandiflora with sizes of 1.80kb (cgAUS1) and 1.85kb (cgAUS2a, 2b), encoding for proteins of 68-69kDa, respectively. cgAUS1 is preferably expressed in young petals indicating a specific role in pigment formation. The 58.9kDa AUS1 holoproenzyme, was recombinantly expressed in E. coli and purified to homogeneity. The enzyme shows only diphenolase activity, catalyzing the conversion of chalcones to aurones and was characterized by SDS-PAGE and shot-gun type nanoUHPLC-ESI-MS/MS.

Purification and characterization of tyrosinase from walnut leaves (Juglans regia).

Polyphenol oxidase (PPO) is a type-3 copper enzyme catalyzing the oxidation of phenolic compounds to their quinone derivates, which are further converted to melanin, a ubiquitous pigment in living organisms. In this study a plant originated tyrosinase was isolated from walnut leaves (Juglans regia) and biochemically characterized. It was possible to isolate and purify the enzyme by means of an aqueous two-phase extraction method followed by chromatographic purification and identification. Interestingly, the enzyme showed a rather high monophenolase activity considering that the main part of plant PPOs with some exceptions solely possess diphenolase activity. The average molecular mass of 39,047 Da (Asp(101)→Arg(445)) was determined very accurately by high resolution mass spectrometry. This proteolytically activated tyrosinase species was identified as a polyphenol oxidase corresponding to the known jrPPO1 sequence by peptide sequencing applying nanoUHPLC-ESI-MS/MS. The polypeptide backbone with sequence coverage of 96% was determined to start from Asp(101) and not to exceed Arg(445).

High level protein-purification allows the unambiguous polypeptide determination of latent isoform PPO4 of mushroom tyrosinase.

Tyrosinases catalyze two initial reaction steps in the formation of melanin. Purification of tyrosinases had always been a process accompanied with various problems caused by enzymatic browning processes. Here, an approach is presented for the purification of the latent enzyme from mushrooms which averts and removes interfering compounds (e.g. polyphenols) in advance to the extraction process. The described method is supposed being well suitable as a general protein purification protocol from natural sources like fungi and plants. The purified enzyme was investigated in detail by means of mass spectrometry: its intact protein mass was determined as 64,247.3 Da and it was identified as number four of in total six isoforms (PPO1-6) by means of sequence analysis. Some PTMs, strain specific sequence disparities and several cleavage sites including the one causing enzyme-activation (Ser³8³) were determined, thus, providing insights on the maturation process of this latent tyrosinase zymogen. Based on these sequence data it can be concluded that the polypeptide backbone of the latent form of the tyrosinase PPO4 ranges from Ser² to Thr565, missing when compared to the gene-derived sequence a small part (46 amino acids) of the C-terminal tail. The high content on hydrophobic amino acids within this missing tail gives rise to speculations whether this part might have a function as a membrane anchor.

Voriconazole pharmacokinetics and safety in immunocompromised children compared to adult patients.

The aim of this study was to investigate the pharmacokinetics and safety of voriconazole after intravenous (i.v.) administration in immunocompromised children (2 to 11 years old) and adults (20 to 60 years old) who required treatment for the prevention or therapy of systemic fungal infections. Nine pediatric patients were treated with a dose of 7 mg/kg i.v. every 12 h for a period of 10 days. Three children and 12 adults received two loading doses of 6 mg/kg i.v. every 12 h, followed by a maintenance dose of 5 mg/kg (children) or 4 mg/kg (adults) twice a day during the entire study period. Trough voriconazole levels in blood over 10 days of therapy and regular voriconazole levels in blood for up to 12 h postdose on day 3 were examined. Wide intra- and interindividual variations in plasma voriconazole levels were noted in each dose group and were most pronounced in the children receiving the 7-mg/kg dose. Five (56%) of them frequently had trough voriconazole levels in plasma below 1 microg/ml or above 6 microg/ml. The recommended dose of 7 mg/kg i.v. in children provides exposure (area under the concentration-time curve) comparable to that observed in adults receiving 4 mg/kg i.v. The children had significantly higher C(max) values; other pharmacokinetic parameters were not significantly different from those of adults. Voriconazole exhibits nonlinear pharmacokinetics in the majority of children. Voriconazole therapy was safe and well tolerated in pediatric and adult patients. The European Medicines Agency-approved i.v. dose of 7 mg/kg can be recommended for children aged 2 to <12 years.

Determination of saliva trough levels for monitoring voriconazole therapy in immunocompromised children and adults.

To evaluate the reliability and practical use of saliva for therapeutic drug monitoring of the antifungal agent voriconazole in immunocompromised patients, a paired-sample study was conducted. Plasma and saliva trough levels were measured in seven children and nine adults who required treatment for the prevention or therapy of systemic fungal infections. The pediatric patients received a voriconazole dosage of 7 mg/kg intravenously twice a day. Adults were treated with two loading doses of 6 mg/kg intravenously followed by a maintenance dose of 4 mg/kg intravenously twice a day. Based on 104 paired plasma/saliva specimens, we found a significant correlation between the voriconazole concentrations in blood and saliva (r > 0.95). The median saliva/plasma voriconazole concentration ratio was 0.34 in children and 0.40 in adults. Intra- and interpatient variability in the saliva/plasma ratios were 22% and 23% in children and 16% and 24% in adults, respectively. Thirty-three percent of plasma trough levels were below 1.0 microg/mL or above 6.0 microg/mL and occurred in six pediatric and four adult patients. Monitoring of salivary concentrations proved to be a realistic alternative in patients when blood drawing is difficult. Especially in therapeutic drug monitoring, an easier sample collection being noninvasive and painless is more acceptable to patients, particularly children.

Determination of voriconazole in human plasma and saliva using high-performance liquid chromatography with fluorescence detection.

Voriconazole is a widely used triazole antifungal agent with a broad spectrum including Aspergillus species. A simple, sensitive and selective high-performance liquid chromatography method for the determination of voriconazole in human plasma and saliva was developed. Drug and internal standard (UK-115 794) were extracted from alkaline plasma and saliva with n-hexane-ethyl acetate (3:1, v/v) and analyzed on a Luna C 18 column with fluorimetric detection set at excitation and emission wavelengths of 254 and 372 nm, respectively. The calibration curve was linear through the range of 0.1-10 microg/ml using a 0.3 ml sample volume. The intra- and inter-day precisions were all below 6.1% for plasma and below 9.1% for saliva. Accuracies ranged from 94 to 109% for both matrices. Mean recovery was 86+/-4% for voriconazole. The method showed acceptable values for precision, recovery and sensitivity and is well suited for routine analysis work and for pharmacokinetic studies.

Rapid and simple method for the analysis of nateglinide in human plasma using HPLC analysis with UV detection.

Nateglinide (NA) is a novel oral mealtime glucose regulator, recently approved for the treatment of type II diabetes mellitus. To facilitate clinical studies investigating the dependence of NA elimination on the genotype of cytochrome P450 isoenzymes, we developed a rapid HPLC method for determination of NA in human plasma samples. The validated limit of quantitation (LOQ) of 0.1 microg/ml is low enough to allow determination of pharmacokinetic parameters of the substance. The intra-assay coefficients of variation (CV) ranged from 1.6 to 12.9% at NA concentrations of 0.5-7.5 microg/ml. The inter-assay variation for the same plasma concentrations ranged from 3.8 to 8.4%. The calibration was linear in the range of 0.1-20 microg/ml. For the quantitation of NA, only 50 microl of plasma were needed. Following protein precipitation in human plasma, the samples were separated by isocratic reversed phase HLPC and analyzed using ultraviolet detection at 210 nm. Sample preparation time and analysis time are both short and allow rapid analysis of large sample sets.