Detection of metallothionein isoforms from three different species using on-line capillary electrophoresis-mass spectrometry

The Del allele of the apolipoprotein B (apoB) signal peptide (SP) insertion/deletion (Ins/Del) polymorphism has been shown to be associated with elevated plasma levels of apoB, cholesterol and low density lipoprotein. It was the aim of the present study to analyse the relation of this gene variation to the risk of coronary artery disease (CAD) and of myocardial infarction (MI) in a population of 2259 male Caucasians, whose coronary anatomy was defined by means of coronary angiography. ApoB SP DelDel genotypes had significantly higher apoB plasma concentrations than InsIns homozygotes (P = 0.0001) and InsDel heterozygotes (P = 0.002); however, the apoB plasma levels of InsIns and InsDel genotypes were essentially the same (P = 0.54). Similar observations were made with respect to ApoB SP genotype-dependent cholesterol plasma concentrations. Since the apoB plasma level was not only associated with the apoB SP Ins/Del gene variation but also to the extent of coronary artery disease (P <0.0001), individuals with an InsIns genotype and without CAD had the lowest and subjects with a DelDel genotype and triple vessel disease the highest apoB plasma levels (P <0.0001). An association of the apoB SP Ins/Del gene variation with CAD was not detected, neither in the total population nor in low risk groups. In contrast, the gene variation was associated with MI (P <0.05). An Odds ratio of 1.18 (95% CI, 1.01-1.39) associated with the Del allele was detected in the total sample (P <0.02). In a subpopulation of individuals with low plasma triglyceride levels ( <154 mg/dl; mean value) and an DD genotype of the angiotensin I-converting enzyme insertion/deletion gene polymorphism an Odds ratio of 2.01 (1.42-3.05) was calculated (P <0.001). The present study presents evidence for a statistically significant difference in the development of MI between genotype classes of the apoB SP Ins/Del gene polymorphism.     

Direct analysis of protein mixtures by tandem mass spectrometry

Methods to identify proteins contained in mixtures are described. The approach uses microcolumn liquid chromatography and automated tandem mass spectrometry in conjunction with protein and nucleotide database searching algorithms. This approach is applied to the identification of proteins obtained by immunoprecipitation reactions, interaction with a GST protein fusion products and interaction with a macromolecular complex.     

Investigation of beta-amyloid peptide by on-line high-performance liquid chromatography coupled to electrospray ionization mass spectrometry

beta(1-39) amyloid peptide is one of the components of the cerebral amyloid deposits that are characteristic of Alzheimer's disease. Solid-phase synthesis of this peptide resulted in a fairly complex crude product containing both the target peptide and a number of side products. High-performance liquid chromatography coupled to electrospray ionization mass spectrometry allowed rapid and reliable identification of both the desired peptide and most of the side products which were found to have relative molecular masses above and below that of the target peptide.     

Tandem mass spectrometry analysis of synthetic opioid peptide analogs

Five synthetic opioid peptides that were designed to have specific opioid receptor-binding properties were studied by low energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). The MS/MS data are required for the analysis of those peptides in ovine plasma in a study to determine the placental transfer of the peptide to the fetus. The synthetic enkephalin-related peptides were: Tyr-D-Arg-Phe-Lys-NH2, (DALDA), N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, (ICI 174,864), Tyr-D-Thr-Gly-Phe-Leu-Thr, (DTLET), Tyr-D-Pen-Gly-Phe-D-Pen-OH, (DPDPE), and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, (CTAP). Liquid secondary ion mass spectrometry (LSIMS) was used for sample desorption-ionization, and a hybrid (E1BE2qQ) tandem mass spectrometer was used to collect the product-ion spectra. A protonated molecule ion, [M + H]+, was observed for each peptide. Amino acid sequence-determining fragment ion were produced by CID and collected by MS/MS for the three linear peptides, and also for the two disulfide-bond-containing peptides in their unreduced and dithiothreitol (DTT)-reduced forms. The detection level for the [M + H]+ ion of DTLET was ca. 3 pmol; and the stabilities of the CTAP and ICI analogs in plasma were studied.     

Rapid peptide mapping by reversed-phase liquid chromatography on nonporous silica with on-line electrospray time-of-flight mass spectrometry

Reversed-phase liquid chromatography (LC) using a nonporous silica support has been combined with electrospray (ES) time-of-flight (TOF) mass spectrometry (MS) for the fast separation and mass detection of peptides. Using this LC method, the resolution of a peptide mixture can be completed is less than 35 s. The resulting chromatographic peak widths are less than 1 s wide. Because of the unique nature of a TOF mass analyzer, complete mass spectra can be acquired at a rate which is sufficient to sample these narrow peaks. When compared with conventional LC, the same separation takes nearly 20 min to complete, and the signal-to-noise ratio observed in the total ion chromatogram is dramatically lower due to the influence of increased background noise in the mass spectra. The limit of detection for a low molecular weight peptide, Val-Pro-Leu, was found to be 6 pmol with the total ion chromatogram and 500 fmol with the reconstructed ion chromatogram. A peptide map of horse heart myoglobin, completed in 3.5 min, is shown as an example of the results which can be obtained from combining this fast LC method with fast ES/TOF/MS detection capability.     

An automated liquid chromatography/mass spectrometry system coupled on-line with microdialysis for the in vivo analysis of contrast agents

An automated high-performance liquid chromatography (HPLC)/mass spectrometry instrument coupled on-line with a microdialysis probe is described for the analysis of contrast agents in biological fluids. This system is used for pharmacokinetic and structure elucidation studies of contrast agents used in X-ray and magnetic resonance imaging investigations. Additionally an LC/MS system for the direct analysis of urine, that obviates the need for conventional sample clean-up, is described.     

ESI/ion trap/ion mobility/time-of-flight mass spectrometry for rapid and sensitive analysis of biomolecular mixtures

An ion trap/ion mobility/time-of-flight mass spectrometry technique is shown to be a rapid and sensitive means of analyzing peptide/protein mixtures. In this approach, an ion trap is used to accumulate ions that have been electrosprayed from a mixture into concentrated packets. The ion packets are injected into a drift tube where components of the mixture are separated based on differences in mobility through a buffer gas. Ions that exit the drift tube are dispersed in a time-of-flight mass spectrometer for mass-to-charge (m/z) determination. The gas-phase separation strategy reduces congestion in the mass spectrum, and experimental mobilities complement m/z measurements in assigning peaks. Examples of the application of the approach to identification of peptides (from tryptic digests) and to separation of charge-state distributions from electrospray of a mixture containing ubiquitin and myoglobin are presented. Most peptides that are observed from tryptic digests of proteins such as cytochrome c and myoglobin can be identified from data that are acquired in under 1 min; studies of mixtures with known compositions indicate that detection limits are approximately 0.5-3 pmol for individual components. Factors that may influence the distributions that are observed, such as storage time in the trap, injection voltages used for the mobility experiment, and variations in ion cross section with charge state, are discussed.     

控冷温度命中率现场判定系统研发

为了实现控冷温度命中率的自动统计,在武钢热轧总厂中厚板分厂现有的轧后控冷系统基础上,研发了温度命中率现场判定及网络查询系统。该系统采用OPC技术现场采集数据,把现场数据计算加工后,上传到本厂的数据中心。通过架设Web服务器,对数据中心的数据进行sql查询来实现网络查询功能。此系统提高了对于控冷产品的质量监控和追溯管理,便于工艺人员根据统计数据改进工艺。     

Automated on-line dialysis for sample preparation and HPLC analysis of antidepressant drugs in human plasma. Inhibition of interaction with the dialysis membrane

Antidepressant drugs interact with the dialysis membrane and were selected as model substances to study inhibition of analyte-membrane interactions. A chemometric approach based on response surface modelling was used for screening and optimisation of dialysis recoveries. Optimal dialysis recoveries (52-65%) were obtained for the model compounds (mianserine, amitriptyline, nortriptyline, imipramine and desimipramine) when a cationic surfactant added to the donor solution of the dialyser was used to inhibit analyte-membrane interactions. Automated analysis of antidepressants in plasma was performed by connecting the ASTED (Automated Sequential Trace Enrichment of Dialysates) system to high-performance liquid chromatography (HPLC). The drugs were detected by ultraviolet detection and fluorescence detection after post-column photochemical reaction. Validation of the method showed linear standard curves for all the drugs in the concentration range 50-2000 nmol 1-1. Within-and between day relative standard deviations ranged from 1.1 to 5.7%.     

棒材在线计数系统的研制

设计了一套棒材在线计数系统，其中的预处理区、计数丝杠、光电传感器以及同步落料机构解决了棒材排列不均匀、计数精度低、无法自动分离等重大技术难题，实现了棒材的在线自动计数。     

Systematic functional analysis of the yeast genome

The genome sequence of the yeast Saccharomyces cerevisiae has provided the first complete inventory of the working parts of a eukaryotic cell. The challenge is now to discover what each of the gene products does and how they interact in a living yeast cell. Systematic and comprehensive approaches to the elucidation of yeast gene function are discussed and the prospects for the functional genomics of eukaryotic organisms evaluated.     

Capillary electrochromatography of biomolecules with on-line electrospray ionization and time-of-flight mass spectrometry

Capillary electrochromatography (CEC) is considered a hybrid of liquid chromatography and capillary electrophoresis. It is expected to combine the high peak efficiency of capillary zone electrophoresis with the versatility and loading capacity of HPLC to bring about another high-performance MS-compatible chromatographic system. This paper explores the potential of CEC coupled with the electrospray ionization and time-of-flight mass spectrometry in biochemical analysis. The packed columns used in this study were tapered at the outlet to retain the packing material, thereby obviating the need for an outlet frit. Electrosmotically driven solvent gradients were employed for the separation of phenylthiohydantoin (PTH)-amino acids by reversed-phase chromatography, and a time-of-flight (TOF) mass spectrometer was employed as the detector for the CEC column effluent. The effect of CEC operating parameters, such as gradient shape, column length, and electric field, on the analytical results from the separation and MS detection of a standard mixture of PTH-amino acids was investigated. Particular attention was paid to the effect of sheath flow-rate, sheath composition and mass spectra acquisition rate on the performance of the electrospray TOF-MS.     

Structural analysis of peptide substrates for mucin-type O-glycosylation

The structures of three nine-residue peptide substrates that show differential kinetics of O-linked glycosylation catalyzed by distinct recombinant uridine diphosphate-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) were investigated by NMR spectroscopy. A combined use of NMR data, molecular modeling techniques, and kinetic data may explain some structural features required for O-glycosylation of these substrates by two GalNAc transferases, GalNAc-T1 and GalNAc-T3. In the proposed model, the formation of an extended backbone structure at the threonine residue to be glycosylated is likely to enhance the O-glycosylation process. The segment of extended structure includes the reactive residue in a beta-like or an inverse gamma-turn conformation and flanking residues in a beta-strand conformation. The hydroxyl group of the threonine to be glycosylated is exposed to solvent, and both the amide proton and carbonyl oxygen of the peptide backbone are exposed to solvent. The exchange rate of the amide proton for the reactive threonine correlated well with substrate efficiency, leading us to hypothesize that this proton may serve as a donor for hydrogen bonding with the active site of the enzyme. The oxygens of the residue to be glycosylated and several flanking residues may also be involved in a set of hydrogen bonds with the GalNAc-T1 and -T3 transferases.     

Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins

Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.     

Automated on-line sample pretreatment system for the determination of trace metals in biological samples by inductively coupled plasma mass spectrometry

A fully automated on-line sample pretreatment system combining microwave digestion with sample preconcentration/matrix separation for the determination of trace metals (Fe, Ni, Cu, Zn, and Pb) in blood and serum samples by inductively coupled plasma mass spectrometry (ICPMS) was designed and evaluated. The samples were mixed with an appropriate reagent and digested in a flow-through, focused microwave-heated oven. After digestion, the sample solution was transferred on-line to a column packed with iminodiacetate-based resin for separation of matrix elements like Na, K, Ca, Mg, Cl, P, and S that might interfere with the measurement by ICPMS. The analytes chelated on the resin were subsequently eluted and led to ICPMS for multielement determination. The whole sample pretreatment process was automatically controlled by a self-designed expert system. The analytical reliability of data from this on-line system was confirmed to be good with the analysis of SRM samples (Seronorm Whole Blood and NIST SRM 1598 Bovine Serum), and the limits of detection (3 sigma) for Fe, Ni, Cu, Zn, and Pb were 68, 0.34, 3.5, 13.4, and 0.22 microgram/L, respectively. With this fully automated on-line system, the determination of analytes in biological fluid samples down to micrograms-per-liter levels has been proven to be feasible, and the sample throughput can achieve up to 6 samples/h.     

Sodium dodecyl sulphate removal from tryptic digest samples for on-line capillary liquid chromatography/electrospray mass spectrometry

An on-line microcolumn switching method was developed for the removal of sodium dodecyl sulphate (SDS) from tryptic digest samples. The system includes two micro-precolumns: a specific ionic detergent trapping column and a preconcentration column. Characterization of the proteinaceous samples, after isolation from the SDS, was performed by capillary liquid chromatography (LC) with UV absorption detection and electrospay mass spectrometry (ESI-MS). Loading and clean-up of the samples and regeneration of the detergent trapping column were performed at 50 microl min(-1), resulting in sample clean-up times of only 30 s. SDS-containing tryptic digested protein samples were directly applied to the micro-precolumns without any previous sample pretreatment. The developed microcolumn switching method permits the on-line analysis of small tryptic digest samples by capillary LC/ESI-MS in the presence of SDS. The method is completely automated and can be performed unattended. The maximum amount of SDS, in terms of loadability and breakthrough, were determined. Also studied were the selection of the loading and clean-up solvents and the recovery of the peptides. Chromatographic separations and mass spectral data confirmed the removal of SDS.