On-line Electrospray Ionization Research Articles

Structural Characterization of BAX 855, a PEGylated Recombinant FVIII

Abstract 4356Baxter and Nektar have developed a longer acting recombinant FVIII (BAX 855), which is manufactured by coupling stable PEG using Nektar technology to Baxter's full-length rFVIII bulk drug substance from the protein-free ADVATE™ manufacturing process. BAX 855 was characterized by a number of analytical methods, focusing on the elucidation of the primary structure, posttranslational modifications, PEGylation site distribution and three-dimensional structure.The primary structure of BAX 855 was investigated using a peptide mapping approach. Samples were digested with trypsin and the resulting peptides were separated by reversed phase chromatography and detected with on-line electrospray ionization mass spectrometry. This approach resulted in a sequence coverage of 94% with good consistency demonstrated between different BAX 855 batches. Protein-bound oligosaccharides were determined by normal phase HPLC of fluorescence labeled N-glycans released by PNGase F treatment. The composition of the N-linked oligosaccharides showed a similar pattern between BAX 855 and unmodified rFVIII, confirming that the N-glycosylation pattern remained intact during the PEGylation process.PEGylation site distribution and detailed analysis of the consistency of PEGylation was investigated by activating BAX 855 with thrombin. The resulting PEGylated and non-PEGylated fragments were separated using a RP-HPLC approach and the bound PEG was measured for each thrombin fragment. The distribution of PEG among the different thrombin fragments of rFVIII was shown to be consistent between several BAX 855 batches. Moreover, RP-HPLC of native BAX 855 showed reproducible subunit composition and PEGylation.SDS-PAGE and Western blot analysis of BAX 855 revealed changes in the electrophoretic pattern due to PEGylation without the appearance of any relevant degradation products. The random PEGylation of rFVIII was shown using antibodies with different epitope specificities. Dynamic light scattering and Fourier-transformed infrared spectroscopy (FTIR) were used to monitor the consistency of three dimensional structures. The mean hydrodynamic diameter of BAX 855 was between 30 and 40 nm, which is a characteristic size for a ∼300-kDa protein. Several BAX 855 batches showed almost overlapping FTIR absorbance spectra, especially in the conformationally susceptible amide I and amide II peak at 1650 and 1550 cm−1, respectively, indicative for good consistency of the manufacturing process.In summary, BAX 855, a PEGylated rFVIII derivative, can be manufactured reproducibly without changes to the protein structure characteristic for a fully functional FVIII molecule. Disclosures:Kaliwoda:Baxter Innovations GmbH: Employment. Graninger:Baxter Innovations GmbH: Employment. Matthiessen:Baxter Innovations GmbH: Employment. Schrenk:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Mitterer:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Spectral trees as a robust annotation tool in LC–MS based metabolomics

The identification of large series of metabolites detectable by mass spectrometry (MS) in crude extracts is a challenging task. In order to test and apply the so-called multistage mass spectrometry (MS n ) spectral tree approach as tool in metabolite identification in complex sample extracts, we firstly performed liquid chromatography (LC) with online electrospray ionization (ESI)–MS n , using crude extracts from both tomato fruit and Arabidopsis leaf. Secondly, the extracts were automatically fractionated by a NanoMate LC-fraction collector/injection robot (Advion) and selected LC-fractions were subsequently analyzed using nanospray-direct infusion to generate offline in-depth MS n spectral trees at high mass resolution. Characterization and subsequent annotation of metabolites was achieved by detailed analysis of the MS n spectral trees, thereby focusing on two major plant secondary metabolite classes: phenolics and glucosinolates. Following this approach, we were able to discriminate all selected flavonoid glycosides, based on their unique MS n fragmentation patterns in either negative or positive ionization mode. As a proof of principle, we report here 127 annotated metabolites in the tomato and Arabidopsis extracts, including 21 novel metabolites. Our results indicate that online LC–MS n fragmentation in combination with databases of in-depth spectral trees generated offline can provide a fast and reliable characterization and annotation of metabolites present in complex crude extracts such as those from plants.

Lipid Peroxidation Modification of Protein Generates Nϵ-(4-Oxononanoyl)lysine as a Pro-inflammatory Ligand

4-Oxo-2(E)-nonenal (ONE), a peroxidation product of ω-6 polyunsaturated fatty acids, covalently reacts with lysine residues to generate a 4-ketoamide-type ONE-lysine adduct, N(ε)-(4-oxononanoyl)lysine (ONL). Using an ONL-coupled protein as the immunogen, we raised the monoclonal antibody (mAb) 9K3 directed to the ONL and conclusively demonstrated that the ONL was produced during the oxidative modification of a low density lipoprotein (LDL) in vitro. In addition, we observed that the ONL was present in atherosclerotic lesions, in which an intense immunoreactivity was mainly localized in the vascular endothelial cells and macrophage- and vascular smooth muscle cell-derived foam cells. Using liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for quantification of the ONL and confirmed that the ONL was indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. To evaluate the biological implications for ONL formation, we examined the recognition of ONL by the scavenger receptor lectin-like oxidized LDL receptor-1 (LOX-1). Using CHO cells stably expressing LOX-1, we evaluated the ability of ONL to compete with the acetylated LDL and found that both the ONE-modified and ONL-coupled proteins inhibited the binding and uptake of the modified LDL. In addition, we demonstrated that the ONL-coupled protein was incorporated into differentiated THP-1 cells via LOX-1. Finally, we examined the effect of ONL on the expression of the inflammation-associated gene in THP-1 and observed that the ONL-coupled proteins significantly induced the expression of atherogenesis-related genes, such as the monocyte chemoattractant protein-1 and tumor necrosis factor-α, in a LOX-1-dependent manner. Thus, ONL was identified to be a potential endogenous ligand for LOX-1.

Determination of amino acids in rat brain microdialysate with 1,2,5,6-dibenzocarbazole-9-ethyl chloroformate as labeling reagent by high performance liquid chromatographic fluorescence detection and mass spectrometric identification

A high performance liquid chromatography method for the determination of 20 amino acids (AAs), using 1,2,5,6-dibenzocarbazole-9-ethyl chloroformate (DBCEOC–Cl) as a novel fluorescent labeling reagent, has been developed and applied for the analysis of AAs in rat brain microdialysate. The simultaneous separation of 20 AA derivatives was achieved on a Hypersil BDS C 18 column with gradient elution. And the identification of AA derivatives was carried out by on-line electrospray ionization mass spectrometry in positive ion mode. The AA derivatives were detected with excitation and emission at 300 nm and 395 nm, respectively. Excellent linear responses were observed with coefficients of >0.9988. The detection limits ranged from 0.217 to 4.75 nmol/L, at a signal-to-noise ratio of 3. The intra-day and inter-day precision for each AA was <3.2% and <4.3%, respectively. The mean recoveries for all AAs studied were in the range of 92.5–105.6%. Good compositional data could be obtained from as little as 15 μL of microdialysate. Facile DBCEOC–Cl derivatization coupled with high performance liquid chromatography and fluorescence detection allowed the development of a highly sensitive method for the quantitative analysis of trace levels of AAs from microdialysate.

Electrochemistry-electrospray ionization FT ICR mass spectrometry (EC ESI MS) of guanine–tyrosine and guanine–glutathione crosslinks formed on-line

Crosslinking of guanine with tyrosine and glutathione is studied by on-line electrochemistry electrospray ionization mass spectrometry (EC ESI MS) in the positive ion mode with a high resolution FT ICR mass spectrometer without the use of an external oxidizer in the sample. Dimeric adducts of guanine with tyrosine ( m/ z 331) and ( m/ z 333) and a dimer of guanine with glutathione ( m/ z 459) are detected in the mass spectra of guanine–tyrosine and guanine–glutathione mixtures in addition to dimers of guanine ( m/ z 303) and tyrosine ( m/ z 361) and ( m/ z 363) and glutathione dimers ( m/ z 613) and ( m/ z 615). Guanine tetramers ( m/ z 627) and tetramers of guanine with 1 or 2 tyrosines, but not with three tyrosines, were also identified in the mass spectra of guanine–tyrosine mixtures. Formation of radicals and other oxidation products during positive ion mode ESI can drive the formation of covalent dimer adducts of guanine with tyrosine and glutathione. When low EC cell voltage is applied in on-line EC ESI MS, changes in ion intensities reflect changes in the oxidation conditions and are more apparent for 2e, 2H + than for the 1e, 1H + oxidation processes. Efficient oxidation during ESI is indicated for analytes with low redox potentials.

Comprehensive proteome analysis using quantitative proteomic technologies

With the completion of genome sequencing of several organisms, attention has been focused to determine the function and functional network of proteins by proteome analysis. The recent techniques of proteomics have been advanced quickly so that the high-throughput and systematic analyses of cellular proteins are enabled in combination with bioinformatics tools. Furthermore, the development of proteomic techniques helps to elucidate the functions of proteins under stress or diseased condition, resulting in the discovery of biomarkers responsible for the biological stimuli. Ultimate goal of proteomics orients toward the entire proteome of life, subcellular localization, biochemical activities, and their regulation. Comprehensive analysis strategies of proteomics can be classified as three categories: (i) protein separation by 2-dimensional gel electrophoresis (2-DE) or liquid chromatography (LC), (ii) protein identification by either Edman sequencing or mass spectrometry (MS), and (iii) quanitation of proteome. Currently MS-based proteomics turns shiftly from qualitative proteome analysis by 2-DE or 2D-LC coupled with off-line matrix assisted laser desorption ionization (MALDI) and on-line electrospray ionization (ESI) MS, respectively, to quantitative proteome analysis. Some new techniques which include top-down mass spectrometry and tandem affinity purification have emerged. The in vitro quantitative proteomic techniques include differential gel electrophoresis with fluorescence dyes, protein-labeling tagging with isotope-coded affinity tag, and peptide-labeling tagging with isobaric tags for relative and absolute quantitation. In addition, stable isotope labeled amino acid can be in vivo labeled into live culture cells through metabolic incorporation. MS-based proteomics extends to detect the phosphopeptide mapping of biologically crucial protein known as one of post-translational modification. These complementary proteomic techniques contribute to not only the understanding of basic biological function but also the application to the applied sciences for industry.

Microfluidic Cell Culture and Metabolism Detection with Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometer

A novel method for the characterization of drug metabolites was developed by integrating chip-based solid-phase extraction (SPE) with an online electrospray ionization quadrupole time-of-fight mass spectrometer (ESI-Q-TOF-MS). The integrated microfluidic device was composed of circular chambers for cell culture and straight microchannels with shrink ends to pack the solid-phase material for sample cleanup and concentration prior to mass analysis. By connecting the two separated microchannels with polyethylene tubes, drug metabolism studies related to functional units, including cell culture, metabolism generation, sample pretreatment, and detection, were all integrated into the microfluidic device. To verify the feasibility of a drug metabolism study on the microfluidic device, the metabolism of vitamin E in human lung epithelial A549 cells was studied. The metabolites were successfully detected by online ESI-Q-TOF-MS with high sensitivity and short analysis time (8 min). By integrating several parallel channels, the desalting and concentration process could be simultaneously achieved. The total sample pretreatment time only needed about 15 min, and solvent consumption could be reduced to less than 100 microL. All this demonstrated that the developed microfluidic device could be a potential useful tool for cellular drug metabolism research.

Increased Oxidative Stress in Healthy Children Following an Exercise Program: A Pilot Study

Exercise can induce oxidative stress or an imbalance between reactive oxygen species and cellular antioxidant defenses. We investigated the effect of a real-life exercise program on systemic oxidative stress measured by urinary concentrations of 8-isoprostaglandin F2alpha (8-iso-PGF2 alpha), a noninvasive index of lipid peroxidation, in a well-characterized pediatric group. Healthy but primarily sedentary, 8- to 10-year-old children (n = 6, mean age 8.8 +/- 0.9 years) of equally distributed healthy weight, overweight, and obese categories, participated in a 5-week exercise program (track and field summer camp, 2 hours/day, 1-2 days/week). By using high-performance liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS), we found a significant (p = .028) increase in group mean urinary 8-iso-PGF2 alpha concentration from 8.163 +/- 6.919 ng/mg creatinine pre-exercise program to 32.320 +/- 16.970 ng/mg creatinine post-exercise program. The increase was also measured at each individual level. We found preliminary evidence that pre- and post-exercise program urinary 8-iso-PGF2 alpha concentrations selectively correlated with children's cardiometabolic characteristics and mood. Our results warrant further exploration of the relationships between pre/post-exercise oxidative stress marker 8-iso-PGF2 alpha and cardiometabolic characteristics, exercise habits, eating habits, and mood to determine whether increased post-exercise oxidative stress in healthy children is part of their normal adaptation to exercise or mediator of oxidative injury.

Molecular Control of Reactive Gas Uptake “on Water”

Reactive gas uptake on environmentally realistic aqueous surfaces is expected to be affected by a combination of multiple interactions. This issue is herein explored in experiments where the formation of Me(3)NH(+) on neat and doped water microjets exposed to Me(3)N(g) is monitored within <1 ms by online electrospray ionization mass spectrometry as a function of pH of the bulk liquid (pH(BLK)). Notably, Me(3)N(g) is protonated on the surface of neat water microjets below pH(BLK) approximately 4, rather than at pH(BLK) less than or approximately pK(A)(Me(3)NH(+)) = 9.8 as in bulk water. Me(3)N(g) uptake is significantly enhanced by anionic surfactants and fulvic acid (a surrogate of complex natural organic matter) above pH(BLK) approximately 4, uniformly depressed by cationics (which otherwise counteract FA effects), and unaffected by n-octanol. The direct hydrogen isotope effects associated with enhanced uptake of Me(3)N(g) on H(2)O/D(2)O microjets implicate a process controlled by proton transfer from interfacial donors whose coverage is electrostatically modulated by ionic headgroups. The finding that the combined effect of fulvic acid and tetrabutylammonium bromide closely matches the geometric mean of their separate effects on TMA uptake is evidence of strong dopant interactions.

Lipid Peroxidation Generates Body Odor Component trans-2-Nonenal Covalently Bound to Protein in Vivo

trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis- and trans-N(epsilon)-3-[(hept-1-enyl)-4-hexylpyridinium]lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis- and trans-HHP-lysine and confirmed that the 2-nonenal-lysine adducts were indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand.

Membrane protein structural insights from chemical labeling and mass spectrometry

Membrane proteins play a central role in virtually all biological processes, and they represent important drug targets. Unfortunately, the application of traditional high-resolution methods such as X-ray crystallography and NMR spectroscopy to membrane proteins remains challenging. This article reviews alternative approaches that involve chemical labeling and mass spectrometry (MS) for gaining insights into membrane protein structure, function, and interactions. Hydrogen/deuterium exchange MS represents an interesting avenue for exploring biomolecular conformations and dynamics, but thus far this technique has not been widely adopted for membrane protein studies. The main focus of this article is on the use of labeling agents that introduce covalent modifications in solvent-accessible protein regions. While it is possible to monitor the occurrence of single-site modifications using traditional biochemical methods or optical spectroscopy, the use of MS greatly enhances the scope and potential of this approach because multiple tagging events can be detected in parallel. The traditional bottom-up workflow of these studies involves the digestion of a chemically labeled membrane protein by a specific protease such as trypsin. This is followed by chromatographic separation of the resulting peptides and on-line electrospray ionization MS. The application of tandem MS allows pinpointing the exact locations of chemical modifications. A particularly exciting aspect is the applicability of covalent labeling techniques to membrane protein within their natural lipid environment, or even inside living cells. Some of these concepts will be illustrated using the oxidative labeling of bacteriorhodopsin as an example, but numerous other labeling agents and protein systems are being highlighted as well. It is hoped that this review will stimulate further developments in the characterization of membrane proteins.

Identification of Sophoridine and Its Metabolites in Rat Urine by Liquid Chromatography-Tandem Mass Spectrometry

Sophoridine is an important quinolizidine alkaloid which has wide pharmacological effect including antiarrhythmia, antitumor, immunological enhancement, and immunosuppression. In this work, a rapid, sensitive and dependable liquid chromatographic–electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of sophoridine and its metabolites in rat urine after administration of a single dose of sophoridine (12 mg/kg) by vena caudalis injection. The rat urine samples were purified by use of a C18 solid-phase extraction cartridge, then chromatographically separated by a reversed-phase C18 column (Aichrom ReliAsil C18, 5 μm, 2 mm × 150 mm i.d.) with 70:30(v/v) methanol-0.01% triethylamine solution (adjusted to pH 3.5 with formic acid) as mobile phase and detected by an on-line ESI ion trap MS-MS detector. Identification and structural elucidation of the metabolites were performed by comparing the molecular weights, retention-times, and MS-MS spectra of them with those of the parent drug. As a result, six phase I metabolites of sophoridine (N-oxide metabolite, mono-hydroxylation metabolite, hexa-dehydrogenation metabolite and three di-dehydrogenation metabolites) were identified in rat urine for the first time.

Analysis of phenolic compounds from different morphological parts of Helichrysum devium by liquid chromatography with on‐line UV and electrospray ionization mass spectrometric detection

A simple and rapid method has been used for the screening and identification of the main phenolic compounds from Helichrysum devium using high-performance liquid chromatography with on-line UV and electrospray ionization mass spectrometric detection (LC-DAD/ESI-MS(n)). The total aerial parts and different morphological parts of the plant, namely leaves, flowers and stems, were analyzed separately. A total of 34 compounds present in the methanolic extract from Helichrysum devium were identified or tentatively characterized based on their UV and mass spectra and retention times. Three of these compounds were positively identified by comparison with reference standards. The phenolic compounds included derivatives of quinic acid, O-glycosylated flavonoids, a caffeic acid derivative and a protocatechuic acid derivative. The characteristic loss of 206 Da from malonylcaffeoyl quinic acid was used to confirm the malonyl linkage to the caffeoyl group. This contribution presents one of the first reports on the analysis of phenolic compounds from Helichrysum devium using LC-DAD/ESI-MS(n) and highlights the prominence of quinic acid derivatives as the main group of phenolic compounds present in these extracts. We also provide evidence that the methanolic extract from the flowers was significantly more complex when compared to that of other morphological parts.

Identification and Characterization of Polyphenolic Antioxidants Using On-Line Liquid Chromatography, Electrochemistry, and Electrospray Ionization Tandem Mass Spectrometry

It is demonstrated that electrochemistry (EC) coupled to liquid chromatography (LC) and electrospray ionization tandem mass spectrometry (LC/EC/ESI-MS/MS) can be used to rapidly obtain information about the antioxidant activity (i.e., oxidation potential) and capacity (i.e., amount) of polyphenolic compounds, including catechin, kaempferol, resveratrol, quercetin, and quercetin glucosides. The described on-line LC/EC/ESI-MS/MS method facilitates the detection and characterization of individual antioxidants based on a combination of the obtained m/z values for the antioxidants and their oxidation products, the potential dependences for the ion intensities, and correlations between the retention times in the LC, EC, and MS chromatograms. As these results provide patterns that can be used in rapid screening for antioxidants in complex samples, the method should be a valuable complement to chemical assays commonly used to determine the total antioxidant capacity of samples. It is shown that the antioxidant capacity for a mixture of polyphenolic compounds depends on the redox potential employed in the evaluation, and this should consequently be taken into account when comparing results from different total antioxidant capacity assays. It is also demonstrated that the inherent antioxidant capacities of phenolic compounds increase with an increasing number of hydroxyl groups and that the potential needed to oxidize the remaining hydroxyl groups increases successively upon oxidation of the compound. Unlike chemical assays, which generally do not provide any information about the identities of the compounds on the molecular level, the present screening method can be used to identify individual antioxidants, rank compounds with respect to their ease of oxidation, and to study the antioxidant capacity at any redox potential of interest.

Characterization of the Caenorhabditis elegans UDP-galactopyranose mutase homolog glf-1 reveals an essential role for galactofuranose metabolism in nematode surface coat synthesis

Galactofuranose (Gal(f)), the furanoic form of d-galactose produced by UDP-galactopyranose mutases (UGMs), is present in surface glycans of some prokaryotes and lower eukaryotes. Absence of the Gal(f) biosynthetic pathway in vertebrates and its importance in several pathogens make UGMs attractive drug targets. Since the existence of Gal(f) in nematodes has not been established, we investigated the role of the Caenorhabditis elegans UGM homolog glf-1 in worm development. glf-1 mutants display significant late embryonic and larval lethality, and other phenotypes indicative of defective surface coat synthesis, the glycan-rich outermost layer of the nematode cuticle. The glf homolog from the protozoan Leishmania major partially complements C. elegans glf-1. glf-1 mutants rescued by L. major glf, which behave as glf-1 hypomorphs, display resistance to infection by Microbacterium nematophilum, a pathogen of rhabditid nematodes thought to bind to surface coat glycans. To confirm the presence of Gal(f) in C. elegans, we analyzed C. elegans nucleotide sugar pools using online electrospray ionization-mass spectrometry (ESI-MS). UDP-Gal(f) was detected in wild-type animals while absent in glf-1 deletion mutants. Our data indicate that Gal(f) likely has a pivotal role in maintenance of surface integrity in nematodes, supporting investigation of UGM as a drug target in parasitic species.

Targeted Metabolomic Evaluation of Arginine Methylation and Cardiovascular Risks

We examine the relationship of related posttranslational modification products of arginine methylation and coronary artery disease (CAD) phenotypes. Plasma was isolated from 1011 consecutive consenting subjects undergoing elective diagnostic cardiac catheterization, and future major adverse cardiac events (MACE, including myocardial infarction, stroke, and death) at 3 years were investigated. Plasma levels of asymmetrical dimethylarginine (ADMA, endogenous nitric oxide synthase [NOS] inhibitor), symmetrical dimethylarginine (SDMA, which lacks NOS inhibitory activity), N-mono-methylarginine (MMA, a potent NOS inhibitor), methyl-lysine (Methyl-Lys, an unrelated methylated amino acid), arginine, and its major catabolites (citrulline and ornithine) were quantified simultaneously by stable isotope dilution HPLC with online electrospray ionization tandem mass spectrometry and adjusted for traditional risk factors, C-reactive protein, and estimated creatinine clearance. High SDMA levels (adjusted odds ratio [OR] 1.6, 95%CI, 1.1 to 2.6, P<0.001), low MMA (adjusted OR 0.5, 95%CI 0.4 to 0.8, P=0.007), but not ADMA (adjusted OR 1.3, 95%CI 0.88 to 2.0, P=0.177) were associated with increased prevalence of significantly obstructive CAD. Elevated levels of SDMA (adjusted Hazard Ratio [HR] 2.4, 95%CI 1.2 to 4.6, P=0.009), ADMA (adjusted HR 2.2, 95%CI 1.2 to 4.0, P=0.015), as well as an integrated index of arginine methylation [ArgMI=(ADMA+SDMA)/MMA] (adjusted HR 2.4, 95%CI 1.3 to 4.5, P=0.006) were significant independent predictors of incident MACE. ArgMI was predictive of incident MACE even following adjustments for global arginine bioavailability, particularly within secondary prevention patients. ADMA, SDMA, and the integrated quantification of arginine methylation (in the form of a methylation index) provided independent risk prediction for both significantly obstructive CAD and incident MACE in stable patients undergoing cardiac evaluation. These results suggest that factors beyond direct NOS inhibition contribute to the clinical associations between methylarginines and CAD outcomes.

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.

Effect of salt in the mobile phase on the critical conditions of poly(ethylene glycol) in liquid chromatography-mass spectrometry coupling

The nature of salts introduced in the chromatographic mobile phase to promote on-line electrospray ionization was shown to be a key parameter in the optimization of LCCC-MS couplings, both using non-polar (Si-C18) and polar (Si-NH2) stationary phases. The critical conditions of poly(ethylene glycol), which reflect a compensation process between exclusion and interaction effects, were strongly modified when changing the size of the cation in the eluent. This phenomenon could be attributed to interactions between the cation and water molecules from the mobile phase, in the case of the non-polar stationary phase, giving rise to a salting out effect due to a lowered solvent quality of the eluent; or between the cation and amino-modified silanols of the polar stationary phase, inducing a decrease of the surface adsorptivity. Accordingly, increasing the size of the cation in the mobile phase caused the polymer molecules to be eluted according to the adsorption mode using non-polar adsorbent and according to the exclusion mode using polar stationary phase.

Direct observation of the autophosphorylation of insulin receptor kinase by mass spectrometry

The catalytic and signaling activities of insulin receptor kinase (IRK) are regulated by the autophosphorylation of three tyrosine residues in a cytoplasmic protein-tyrosine kinase domain at Tyro 1158, Tyro 1162 and Tyro 1163. In this study, time-course of the auphosphorylation of the core kinase (residues 978–1283) from IRK was directly investigated by online electrospray ionization mass spectrometry. It is found that two tyrosine residues were phosphorylated in reaction time range of 30 min. This study implies that mass spectrometric technique must be a powerful tool to directly monitor the biological macromolecular modification and will also provide the information of the order and the mechanism of autophosphorylation at the tyrosine sites coupled with tandem mass spectrometric technique.

The catalytic mechanism of indole-3-glycerol phosphate synthase (IGPS) investigated by electrospray ionization (tandem) mass spectrometry

An enzymatic reaction has been monitored by on-line direct infusion electrospray ionization (tandem) mass spectrometry. Using this fast and sensitive technique, a key and transient intermediate of Mycobacterium tuberculosis indole-3-glycerol phosphate synthase (IGPS)-catalyzed reaction has been trapped. The reaction catalyzed by indole-3-glycerol phosphate synthase is part of the tryptophan biosynthetic pathway, and is not present in mammals, including humans. This peculiarity renders this enzyme a potential target for the development of biospecific agents with potential anti-TB activity. The present results indicate the presence of two intermediates in the mechanism of this enzymatic reaction.