Characterization of γ-carboxylated tryptic peptides by collision-induced dissociation and electron transfer dissociation mass spectrometry

Vitamin K-dependent carboxylation of glutamic acid (Glu) residues into γ-carboxyglutamic acid (Gla) is a post-translational modification essential for normal protein activity of, for example, proteins involved in the blood coagulation system. These proteins may contain as many as 12 sites for γ-carboxylation within a protein sequence of 45 amino acid residues. In the biopharmaceutical industry, powerful analytical techniques are required for identification and localization of modified sites. We here present comparatively easy and rapid methods for studies of Gla-containing proteins using recent technology. The performances of two mass spectrometric fragmentation techniques, collision-induced dissociation (CID) and electron transfer dissociation (ETD), were evaluated with respect to γ-carboxylated peptides, applying on-line LC-ion trap MS. ETD MS has so far not been reported for Gla-containing peptides and the applicability of CID for heavily γ-carboxylated proteins has not been evaluated. The anticoagulant protein, protein C, containing nine Gla-sites, was chosen as a model protein. After tryptic digestion, three peptides containing Gla-residues were detected by MS; a 1.2 kDa fragment containing two Gla-residues, a 4.5 kDa peptide containing seven residues and also the 5.6 kDa tryptic peptides containing all nine Gla-residues. Regarding the shortest peptide, both CID and ETD provided extensive peptide sequencing. For the larger peptides, fragmentation by CID resulted in loss of the 44 Da CO(2)-group, while little additional fragmentation of the peptide chain was observed. In contrast, ETD resulted in comprehensive fragmentation of the peptide backbone. The study demonstrates that the combination of both techniques would be beneficial and complementary for investigation of γ-carboxylated proteins and peptides.     

Environmental PAH analysis by gas chromatography–atmospheric pressure laser ionization–time-of-flight–mass spectrometry (GC-APLI-MS)

The application of polycyclic aromatic hydrocarbon (PAH) analysis by gas chromatography coupled with atmospheric pressure laser ionization and mass spectrometry (GC-APLI-MS) to environmental samples was investigated in the study. The limit of detection for 40 PAH in a standard mixture was 5–100 fg, demonstrating GC-APLI-MS to be a highly sensitive technique and more sensitive by a factor of 100–3,500 compared to GC-MS. Acenaphthylene and cyclopenta[cd]pyrene were not detectable <2,500 fg per injection. To make use of this very high PAH sensitivity, the technique was applied to samples of environmental interest with limited available sample amounts such as particulate matter (PM), soot and a sample from a bioaccumulation test with Lumbriculus variegatus. First, special sample preparation was necessary and ultrasonic extraction proved to be suitable, if a thorough clean-up was performed and plastic materials avoided. By GC-APLI-MS and GC-MS, 224 and 28 single PAH compounds were detected in PM, about 1,000 and 15 in birch soot, and 9 and 2 in worm tissue, respectively, revealing the enormous potential of the method. The selectivity of GC-APLI-MS was shown for a crude oil where >2,200 PAH were detected without any sample preparation.     

Multiresidue analysis of 83 pesticides and 12 dioxin-like polychlorinated biphenyls in wine by gas chromatography–time-of-flight mass spectrometry

A multiresidue method is described for simultaneous estimation of 83 pesticides and 12 dioxin-like polychlorinated biphenyls (PCBs) in red and white wines. The samples (20 mL wine, acidified with 20 mL 1% HCl) were extracted with 10 mL ethyl acetate (+20 g sodium sulphate) and cleaned by dispersive solid-phase extraction (DSPE) with anhydrous calcium chloride and Florisil successively. The final extract (5 mL) was solvent exchanged to 1 mL of cyclohexane:ethyl acetate (9:1), further cleaned by DSPE with 25 mg primary secondary amine sorbent and analyzed by gas chromatography–time-of-flight mass spectrometry (GC–TOF-MS) within 31 min run time. The limits of quantification of most analytes were ≤10–20 μg/L. Acidification of wine prior to extraction prevented hydrolysis of organophosphorous pesticides as well as dicofol, whereas treatment with CaCl₂ minimized the fatty acid co-extractives significantly. Solvent exchange to cyclohexane:ethyl acetate (9:1) further minimized the co-extractives. Recoveries at 5, 10 and 20 ng/mL were >80% for most analytes except cyprodinil, buprofezin and iprodione. The expanded uncertainties at 10 ng/mL were <20% for most analytes. Intra-laboratory precision in terms of Horwitz ratio of all the analytes was below 0.5, suggesting ruggedness of the method. Effectively, the method detection limit for most analytes was as low as up to 1 ng/mL in both red and white wine, except for cyfluthrin and cypermethrin.     

Fast counter-electroosmotic capillary electrophoresis–time-of-flight mass spectrometry of hyaluronan oligosaccharides

Fast capillary electrophoresis–mass spectrometry measurements under counter-electroosmotic analyte migration conditions are presented. Efficient separations of a homologous series of six hyaluronan oligosaccharides (comprising 1–6 hyalobiuronic acid moieties) could be completed in 65 s. Separations were achieved in short-length fused silica capillaries under high electric field strengths of up to 1.25 kV·cm⁻¹. Capillary inner diameters ranging from 5 to 50 μm were investigated, resulting in an optimal value of 15 μm. The influence of capillary dimensions and buffer composition on separation efficiency and sensitivity are discussed. Optimal separations were achieved using a 28 cm × 15 μm capillary, a separation high voltage of 35 kV, a background electrolyte of 25 mM ammonium acetate adjusted to pH 8.5, and negative ionization mode. The optimized method was successfully applied to a bovine testicular hyaluronidase digest of hyaluronan. Only minimal sample pretreatment for protein-containing samples is required. The simple manual injection procedure and fast separations allow for a sample throughput of 35 samples per hour.     

Direct elemental analysis of biodiesel by inductively coupled plasma–mass spectrometry

An ICP–MS instrument fitted with an octopole reaction system (ORS) was used to directly measure the inorganic contents of several biofuel materials. Following sample preparation by simple dilution in kerosene, the biofuels were analysed directly. The ORS effectively removed matrix- and plasma-based spectral interferences to enable measurement of all important analytes, including sulfur, at levels below those possible by ICP–OES. A range of commonly produced biofuels was analysed, and spike recovery and long-term stability data was acquired. Suitably configured ICP–MS has been shown to be a fast and very sensitive technique for the elemental analysis of biofuels.     

Identification of polyethanolamines by chromatography–mass spectrometry

Presumable structures of polyethanolamines, synthesized by the catalytic β-hydroxyethylation of ammonia with an excess of ethylene oxide are determined by gas chromatography–electron ionization mass spectrometry and tandem mass spectrometry qualitatively, following fragmentation ways and also taking into account retention data. The preferable paths of consecutive reactions of the synthesis of polyethanolamines in a homologous series of isomers are found.     

Oligosaccharide analysis by graphitized carbon liquid chromatography–mass spectrometry

Structural analysis of complex mixtures of oligosaccharides using tandem mass spectrometry is regularly complicated by the presence of a multitude of structural isomers. Detailed structural analysis is, therefore, often achieved by combining oligosaccharide separation by HPLC with online electrospray ionization and mass spectrometric detection. A very popular and promising method for analysis of oligosaccharides, which is covered by this review, is graphitized carbon HPLC–ESI-MS. The oligosaccharides may be applied in native or reduced form, after labeling with a fluorescent tag, or in the permethylated form. Elution can be accomplished by aqueous organic solvent mixtures containing low concentrations of acids or volatile buffers; this enables online ESI-MS analysis in positive-ion or negative-ion mode. Importantly, graphitized carbon HPLC is often able to resolve many glycan isomers, which may then be analyzed individually by tandem mass spectrometry for structure elucidation. While graphitized carbon HPLC–MS for glycan analysis is still only applied by a limited number of groups, more users are expected to apply this method when databases which support structural assignment become available.

Carbohydrate analysis of hemicelluloses by gas chromatography–mass spectrometry of acetylated methyl glycosides

A method based on gas chromatography-mass spectrometry analysis of acetylated methyl glycosides was developed in order to analyze monosaccharides obtained from various hemicelluloses. The derivatives of monosaccharide standards, arabinose, glucose, and xylose were studied in detail and (13)C-labeled analogues were used for identification and quantitative analysis. Excellent chromatographic separation of the monosaccharide derivatives was found and identification of the anomeric configuration was feasible through a prepared and identified pure methyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside. The electron ionization mass spectrum and fragmentation path was studied for each monosaccharide derivative. Fragment ion pairs of labeled and unlabeled monosaccharides were used for quantification; m/z 243/248 for glucose, 128/132 for xylose, and 217/218 for arabinose. Using the intensity ratios obtained from the extracted ion chromatograms, accurate quantification of monosaccharide constituents of selected hemicelluloses was demonstrated.     

Specific screening method for dextran and hydroxyethyl starch in human urine by size exclusion chromatography–in-source collision-induced dissociation–time-of-flight mass spectrometry

The use of plasma volume expanders (PVE), such as dextran (DEX) and hydroxyethyl starch (HES), is prohibited in sports. DEX is a naturally occurring glucose polymer, whereas HES is synthetically produced from amylopectin starch by substitution with hydroxyethyl groups. In doping control, the commonly applied enzymatic and colorimetric screening methods are lacking adequate specificity for DEX and HES. Also, gas chromatographic–mass spectrometic (GC-MS) screening methods have specificity issues with DEX. In addition, due to the nature of the target compounds, time-consuming derivatisation steps are required in GC-MS. Based on the high molecular weight of carbohydrate polymers excreted in urine after administration of DEX and HES, a screening method was developed involving size exclusion chromatography (SEC) combined with time-of-flight mass spectrometry (TOFMS). By using solely a SEC guard column as an analytical column allowed sufficient chromatographic resolution in a minimal amount of time and with reasonable repeatability (average RSD of 10%). Detector response was linear throughout the measurement range with R ² > 0.99 for both analytes. Limits of detection were 100 and 250 μg mL⁻¹ for DEX and HES, respectively. Ion suppression was found to be 52% at maximum. In-source collision-induced dissociation (ISCID) was used to produce characteristic fragments at a mass accuracy better than 2 mDa. The specificity of the SEC–ISCID–TOFMS method was demonstrated with 120 PVE negative doping control samples analyzed in parallel with a routine GC-MS screening method. In addition, seven urine samples from diabetic athletes, causing interpretation problems in routine GC-MS, showed here a definitely negative profile.     

Optimization of antibiotic analysis in water by solid-phase extraction and high performance liquid chromatography–mass spectrometry/mass spectrometry

This paper describes the development of an optimized method based on solid-phase extraction (SPE) followed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) for the simultaneous analysis of ten antibiotic compounds including tetracyclines, sulfonamides, macrolides and quinolones. LC–MS/MS sensitivity has been optimized by alterations to both LC and MS operations. Of the two high resolution columns tested, Waters Symmetry C₁₈ endcapped and Agilent Zorbax Bonus-RP, the latter was found to show better performance in producing sharp peaks and clear separation for most of the target compounds. Optimization of the MS fragmentation collision and cone energy enhanced the peak areas of the target analytes. The recovery of the target compounds from water samples was most efficient on Waters Oasis HLB SPE cartridge, while methanol was shown to be the most suitable solvent for desorbing the compounds from SPE. In addition, acidification of samples prior to SPE was shown to enhance the recovery of the compounds. To ensure a satisfactory recovery, the flow rate through SPE should be maintained at ≤10 mL min⁻¹. The method was successfully applied to the analysis of antibiotics from environmental water samples, with concentrations being <LOD in tap water, between <LOD to 28 ng L⁻¹ in river water and between <LOD to 230 ng L⁻¹ in sewage effluent.     

Study of archaeological artifact by chromatography–mass spectrometry

An archaeological artifact, representing a compound piece of the Bronze Age, the outer surface of which is encrusted with seeds, has been investigated. The chemical composition of the organic binder was determined by gas chromatography and mass spectrometry; X-ray fluorescence analysis was used to determine the nature of the inorganic base of the artifact.     

Identification and determination of mycotoxins and food additives in feed by HPLC–high-resolution time-of-flight mass spectrometry

High-resolution time-of-flight mass spectrometry combined with high performance liquid chromatography is proposed for the detection and determination of 25 mycotoxins and 8 food additives (coccidiostats) in animal feed, using simplified and rapid sample preparation. We developed a procedure for the identification and determination of analytes by the standard addition method. The lower limit of the analytical range is 1 (400) µg/kg for mycotoxins; the analytical range for coccidiostats in feed is 10–200 mg/kg. The relative standard deviation of the results does not exceed 10%. The analysis time is 0.5–1 h.     

Quantitative analysis of highly homologous proteins: the challenge of assaying the “CYP-ome” by mass spectrometry

Cytochrome P450 enzymes comprise families of highly homologous proteins. These proteins play a pivotal role in oxidative drug metabolism and are important targets in drug discovery research. Proteomics today is a valuable tool for the analysis of proteins. In the past, qualitative analysis of the proteome was the main focus of research, but in the last few years interest in the mathematical modelling of protein networks has been growing and so has the demand on quantitative proteome analysis. As a thorough understanding of cytochrome P450 dependent metabolism is crucial for drug discovery, it is thus not astounding that cytochrome P450 enzymes are a target for quantitative proteomics research. In this article, we review the techniques available for quantitative proteome analysis and to what extent these techniques have been used for the quantification of cytochrome P450 enzymes and give a brief outlook of the techniques that have promising potential for the analysis of these proteins in the future.     

Classification of time-of-flight secondary ion mass spectrometry spectra from complex Cu–Fe sulphides by principal component analysis and artificial neural networks

Artificial neural network (ANN) and a hybrid principal component analysis-artificial neural network (PCA-ANN) classifiers have been successfully implemented for classification of static time-of-flight secondary ion mass spectrometry (ToF-SIMS) mass spectra collected from complex Cu–Fe sulphides (chalcopyrite, bornite, chalcocite and pyrite) at different flotation conditions. ANNs are very good pattern classifiers because of: their ability to learn and generalise patterns that are not linearly separable; their fault and noise tolerance capability; and high parallelism. In the first approach, fragments from the whole ToF-SIMS spectrum were used as input to the ANN, the model yielded high overall correct classification rates of 100% for feed samples, 88% for conditioned feed samples and 91% for Eh modified samples. In the second approach, the hybrid pattern classifier PCA-ANN was integrated. PCA is a very effective multivariate data analysis tool applied to enhance species features and reduce data dimensionality. Principal component (PC) scores which accounted for 95% of the raw spectral data variance, were used as input to the ANN, the model yielded high overall correct classification rates of 88% for conditioned feed samples and 95% for Eh modified samples.     

Geographic characterization of Greek wine by inductively coupled plasma–mass spectrometry macroelemental analysis

Abstract

Studying wine mineral profile has been proven as a valuable tool in geographical origin discrimination and authenticity for both producers and consumers. Adulteration of wines, in terms of geographical origin or variety, is considered a major topic of extensive research. Traceability and authenticity of wines have been previously studied on the basis of typical mineral element patterns by means of chemometric methods. In this context, analytical methods were developed for the determination of mineral elements in wines by inductively coupled plasma–mass spectrometry. This study aimed at classifying selected varietal Greek wines from various regions by employing instrumental analysis. Preliminary data of wine mineral content enabled for the classification of samples according to geographical origin and variety. However, further work is required in order to draw more valid conclusions and to obtain a detailed map of the mineral element content of Greek wines according to their geographical origin and/or variety.     

Oxidative degradation products analysis of polymer materials by pyrolysis gas chromatography–mass spectrometry

Pyrolysis gas chromatography–mass spectroscopy (PGC–MS) has been proved to be a powerful method to analyze both the volatile additives and the macromolecular structure of polymer materials. In this paper, flash evaporation technique was used to analyze the volatile degradation products of polymer materials during natural and artificial aging. In high density polyethylene (HDPE) composites, mainly n-alkanes with carbon number from 14 to 29 were detected after natural aging, while no oxidative product was found. Different composites have different n-alkane distributions. In contrast, various oxidative products including ketones, alcohols, esters and unsaturated species could be found in aged polypropylene (PP) nanocomposites. Nanoparticles accelerated the chain scission of PP and increased the formation of oxidative products significantly. During thermal oxidation of nitrile rubber (NBR) seal rubbers, heat/oxidation-induced extra crosslinking predominated and no volatile degradation products was detected. The main change happened in the volatiles is the decrease of additives, especially paraffins, antioxidant RD and hindered phenol. This resulted in the hardening of the rubber and the weakening of the protection from oxidation. Furthermore, the additive distribution along the depth was investigated, showing different migration speeds of different additives. From the additive levels remained in the NBR rubber, it is possible to predict the degradation status. In summary, PGC–MS can supply abundant information of polymer degradation and is helpful for mechanism research.     

Critical evaluation of the potential of radiofrequency pulsed glow discharge–time-of-flight mass spectrometry for depth-profile analysis of innovative materials

The combination of radiofrequency pulsed glow discharge (RF-PGD) analytical plasmas with time-of-flight mass spectrometry (TOFMS) has promoted the applicability of this ion source to direct analysis of innovative materials. In this sense, this emerging technique enables multi-elemental depth profiling with high depth resolution and sensitivity, and simultaneous production of elemental, structural, and molecular information. The analytical potential and trends of this technique are critically presented, including comparison with other complementary and well-established techniques (e.g. SIMS, GD–OES, etc.). An overview of recent applications of RF-PGD–TOFMS is given, including analysis of nano-structured materials, coated-glasses, photovoltaic materials, and polymer coatings     

Residue analysis of glucocorticoids in bovine milk by liquid chromatography–tandem mass spectrometry

A sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 13 steroidal anti-inflammatory drugs in bovine milk is presented. Due to their weakly acid nature, analytes were separated by ion suppression reversed phase chromatography and detected in positive-ion mode by a high flow electrospray source. Dexamethasone-d4 was used as internal standard. The sample preparation was simple and reliable; it included acidic deproteinization of milk followed by sample enrichment and clean-up, utilizing a C18 solid phase extraction cartridge. Recoveries exceeded 70% with an intra-day precision not larger than 12%. The efficiency of the sample clean-up and internal standardization rendered negligible the matrix effect, estimated by comparing standard and matrix-matched calibration curves. A small-scale reconnaissance was carried out on several raw and whole fresh milk samples. A large number of analyzed samples showed a chromatographic peak, in the retention time window of cortisol, at levels included between its decision limit (CCα) and detection capability (CCβ). As a result of a heat-induced transformation, an isomeric product of triamcinolone was observed during the extract evaporation. Since this rearrangement might occur during the milk pasteurization process, LC-MS/MS and ¹H-NMR investigations were performed out to conclusively differentiate the two isomers. One- and two-dimensional proton NMR spectra were able to identify the transformation product as 9a-fluoro-11b,16a-trihydroxy-17b-hydroxymethyl-D-homoandrosta-1,4-diene-3,17a-dione.