A novel approach for identification and measurement of hemoglobin adducts with 1,2,3,4-diepoxybutane by liquid chromatography/electrospray ionisation mass spectrometry and matrix-assisted laser desorption/ionisation tandem mass spectrometry

The structural characterisation of the adducts formed by in vitro interaction of hemoglobin (Hb) with 1,2,3,4-diepoxybutane (DEB), the most reactive 1,3-butadiene (BD) metabolite, was obtained by liquid chromatography/electrospray ionisation mass spectrometry (LC/ES-MS) analysis of modified tryptic peptides of human hemoglobin chains. The reactive sites of human hemoglobin towards DEB and its hydroxylated derivatives (trihydroxybutyl (THB)-derivatives) were identified through the characterisation of alkylated tryptic peptides by matrix-assisted laser desorption/ionisation tandem mass spectrometry (MALDI-MS/MS). Based on this characterisation, a procedure was set up to measure the Hb-adducts of THB-derivatives by isotope dilution mass spectrometry with the use of a deuterated peptide standard. The results obtained here could permit optimisation of molecular dosimetry of BD-adducts, and extension of the analysis to the biological monitoring of occupational exposure to butadiene.     

Coupling of nanoflow liquid chromatography to matrix-assisted laser desorption/ionization mass spectrometry: real-time liquid chromatography run mapping on a MALDI plate

The major obstacle in the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) instruments in the analysis of complex proteome samples is the lack of a direct coupling of a highly resolving separation technique with the mass spectrometer itself. To overcome this drawback, a spotting device for capillary and nanoflow liquid chromatography (LC) with a special liquid deposition principle for lowest volumes was developed. The instrument is able to perform MALDI spotting in real time in order to deposit the LC run on the MALDI plate, and therefore couples the high resolution power of nano-RP-HPLC separation directly with MALDI-MS. This work describes the development and optimization of a method for spotting with online matrix addition, and illustrates its use in the analysis of a complex proteome sample.     

液相基质辅助激光解吸电离-傅里叶变换离子回旋共振质谱研究

选用液相基质制样,考察了激光强度、回旋池开门时间等因数对基质辅助激光解吸电离-傅里叶变换离子回旋共振质谱(MALDI—FT—ICR—MS)检测结果的影响,优化了实验条件。使用液相制样方法对5类实际样品进行了MALDI—FT-ICR—MS检测,结果表明：液相基质具有很好的通用性,质谱信号稳定、持久。利用FT-ICR—MS特有的超高分辨率与准确度,能够很准确地测定化合物组成。     

Enhancing the selectivity of liquid chromatography–mass spectrometry by using trilinear decomposition on LC‐MS data: An application to three‐way calibration of coeluting analytes in human plasma

The high selectivities of liquid chromatography and mass spectrometry make liquid chromatography–mass spectrometry one of the most popular tools for quantitative analysis in complex chemical, biological, and environmental systems, while the potential mathematical selectivity of liquid chromatography–mass spectrometry is rarely investigated. This work discussed the mathematical selectivity of liquid chromatography–mass spectrometry by three‐way calibration based on the trilinear model, with an application to quantitative analysis of coeluting aromatic amino acids in human plasma. By the trilinear decomposition of the constructed liquid chromatography–mass spectrometry‐sample trilinear model and individual regression of the decomposed relative intensity versus concentration, the proposed three‐way calibration method successfully achieved quantitative analysis of coeluting aromatic amino acids in human plasma, even in the presence of uncalibrated interferent(s) and a varying background. This analytical method can ease the requirements for sample preparation and complete chromatographic separation of components, reduce the use of organic solvents, decrease the time of chromatographic separation, and increase the peak capacity of liquid chromatography–mass spectrometry. As a “green analytical method”, the liquid chromatography–mass spectrometry three‐way calibration method can provide a promising tool for direct and fast quantitative analysis in complex systems containing uncalibrated spectral interferents, especially for the situation where the coelution problem is difficult to overcome.     

Tissue sample preparations for preclinical research determined by molecular imaging mass spectrometry using matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization - imaging mass spectrometry is an alternative tool, which can be implemented in order to obtain and visualize the “omic” signature of tissue samples. Its application to clinical study enables simultaneous imaging-based morphological observations and mass spectrometry analysis. Application of fully informative material like tissue allows obtaining the complex and unique profile of analyzed samples. This knowledge leads to diagnosing disease, studying the mechanism of cancer development, selecting the potential biomarkers as well as correlating obtained images with prognosis. Nevertheless, it is worth noticing that this method is found to be objective but the result of the analysis is mainly influenced by the sample preparation protocol, including the collection of biological material, its preservation, and processing. However, the application of this approach requires a special sample preparation procedure. The main goal of the study is to present the current knowledge on the clinical application of matrix-assisted laser desorption/ionization with imaging mass spectrometry in cancer research, with particular emphasis on the sample preparation step. For this purpose, several protocols based on cryosections and formalin-fixed paraffin-embedded tissue were compiled and compared, taking into account the measured metabolites of potential diagnostic importance for a given type of cancer.     

Ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact hemoglobin complex from whole human blood

Ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOFMS) was applied to the analysis of intact human hemoglobin complex directly from whole human blood. The less acidic matrix substance 2,6-dihydroxyacetophenone provided sufficient insensitivity to the impurities present in this crude biological matrix to make any sample pretreatment except dilution dispensable. This matrix facilitated exact molecular mass determination of the non-assembled hemoglobin alpha- and beta-chain (SD < or = +/-0.28 Da and Deltam < or =6.4 ppm for n = 10) if trifluoroacetic acid was used as an additive. Replacement of the denaturing additive trifluoroacetic acid by the neutral salt ammonium acetate allowed the detection of the intact hemoglobin alpha(2)beta(2)H(n)-assembly (n = 0-4) and the alphabeta-subassembly. A prerequisite for the detection of the noncovalent complex was the application of a very soft sample preparation procedure. Crystal morphology, sample concentration and laser pulse energy were also found to be important parameters for the analysis of the intact protein complexes. However, comparison with published electrospray ionization (ESI)-MS results on mammalian hemoglobin molecules shows that, even under the applied gentle conditions, MALDI does not provide a completely reliable picture of the solution-phase equilibrium. In contrast to ESI, especially extensive loss of the heme b (H) groups was noticed. The disruption of the rather stable heme b-globin interaction is assumed to be induced by photo-excitation during the desorption/ionization process.     

Matrix-assisted filament desorption/ionization mass spectrometry

We describe an improved sample preparation method for pulsed filament desorption–ionization mass spectrometry. Samples were deposited in the presence of an excess of liquid or solid matrices. Especially with liquid matrices such as glycerol, this allowed stable and reproducible ion production for a variety of compounds, including biomolecules and synthetic polymers. Substances with molecular weights up to 3000 Da could be desorbed, ionized, and detected by time-of-flight mass spectrometry.     

MALDI-TOF-MS用于组合化学肽库的鉴定

组合化学是一种快速制备大量相关化合物,并可快速对其进行生物活性筛选的革新性的技术和方法。组合化学法自问世以来在生命科学和医药科学技术等领域获得了飞速发展。组合化学肽库已成为同时制备大量不同序列多肽的方法,并已广泛用于抗原决定簇、生物分子相互作用、亲和抑制剂、新药筛选等研究。在亲和筛选研究中,合成组合肽库的评价以及结构鉴定是研究成败的关键步骤之一。     

Covalently bonded aptamer‐functionalised magnetic mesoporous carbon for high‐efficiency chloramphenicol detection

A novel aptamer‐modified magnetic mesoporous carbon was prepared to develop a specific and sensitive magnetic solid‐phase extraction method through combination with ultra‐high performance liquid chromatography‐tandem mass spectrometry for the analysis chloramphenicol in complex samples. More specifically, the chloramphenicol aptamer‐modified Mg/Al layered double hydroxide magnetic mesoporous carbon was employed as a novel magnetic solid‐phase extraction sorbent for analyte enrichment and sample clean‐up. The extraction solvent, extraction time, desorption solvent, and desorption time were investigated. It was found that the mesoporous structure and aptamer‐based affinity interactions resulted in acceptable selective recognition and a good chemical stability toward trace amounts of chloramphenicol. Upon combination with the ultra‐high performance liquid chromatography‐tandem mass spectrometry technique, a specific and sensitive recognition method was developed with a low limit of detection (0.94 pmol/L, S/N = 3) for chloramphenicol analysis. The developed method was successfully employed for the determination of chloramphenicol in complex serum, milk powders, fish and chicken samples, giving recoveries of 87.0‐107% with relative standard deviations of 3.1‐9.7%.     

Rapid screening and determination of designer drugs in saliva by a nib‐assisted paper spray‐mass spectrometry and separation technique

A method for the rapid screening and determination of amphetamine‐type designer drugs in saliva by a novel nib‐assisted paper spray‐mass spectrometry procedure is described. Under optimized conditions, the limit of detections for amphetamine derivatives (model samples: o‐, m‐, p‐chloroamphetamine and o‐, m‐, p‐fluoroamphetamine, respectively) were determined to 0.1 μg/mL by the nib‐assisted paper spray‐mass spectrometry method. This method is easier and has a higher sensitivity than similar methodologies, including atmospheric pressure/matrix‐assisted laser desorption ionization mass spectrometry and electrospray‐assisted laser desorption ionization/mass spectrometry. Data obtained using more classical separation methods, including liquid chromatography and capillary electrophoresis, are also reported.     

MALDI质谱检测蛋白质与富勒醇的非共价复合物

基质辅助激光解吸电离(MALDI)质谱由于受到酸性基质、样品制备、激光诱导聚合和基质加合物的形成等条件的限制而难以用于非共价复合物的检测.本文以芥子酸为基质,观察到蛋白质与富勒醇的特殊相互作用,一些质谱特征,如质量数迁移、宽的加合峰和定量结合比表明,在蛋白质和富勒醇之间形成了特殊的非共价复合物.其中,血红蛋白与富勒醇的结合比是1:4,而肌红蛋白与富勒醇的结合比是1:1.实验结果表明:富勒醇可用来保护血红蛋白,有在酸性介质中防止其分解的作用.因此,通过在基质组份中添加特性有机化合物保护被测样品,有可能实现用MALDI质谱测定四级结构蛋白质的分子量.     

High-resolution field desorption mass spectrometry: Part VII. Explosives and Explosive Mixtures4

Standard explosives and technical mixtures of explosives have been investigated by field desorption mass spectrometry. The compounds investigated gave intense molecular ions or protonated molecules and structurally significant fragmentation. For comparison, the corresponding electron impact and chemical ionization mass spectrometry data are reported. Emission-controlled field desorption, photographic detection, and accurate mass measurements enabled the components of the technical mixtures to be identified. An example of the determination of an additive in a technical product by field desorption mass spectrometry and stable isotope dilution is given. The use of these techniques for quality control of explosives and for forensic investigations appears to be promising.     

Characterization of low molecular weight hydrocarbon oligomers by laser desorption/ionization time-of-flight mass spectrometry using a solvent-free sample preparation method.

A new solvent-free sample preparation method using silver trifluoroacetate (AgTFA) was developed for the analysis of low molecular weight paraffins and microcrystalline waxes by laser desorption/ionization time-of-flight mass spectrometry (LDI-TOFMS). Experiments show that spectral quality can be enhanced by dispersing AgTFA directly in liquid paraffins without the use of additional solvents. This preparation mixture is applied directly to the MALDI probe. Solid waxes could be examined by melting prior to analysis. The method also provides sufficiently reproducible spectra that peak area ratios between mono- and bicyclic alkane peaks indicated variations in the cycloalkane content of paraffin samples. Dehydrogenation of hydrocarbons observed during the desorption/ionization process was studied by analysis of alkane standards.     

Ambient ionisation mass spectrometry for the characterisation of polymers and polymer additives: A review

The purpose of this review is to showcase the present capabilities of ambient sampling and ionisation technologies for the analysis of polymers and polymer additives by mass spectrometry (MS) while simultaneously highlighting their advantages and limitations in a critical fashion. To qualify as an ambient ionisation technique, the method must be able to probe the surface of solid or liquid samples while operating in an open environment, allowing a variety of sample sizes, shapes, and substrate materials to be analysed. The main sections of this review will be guided by the underlying principle governing the desorption/extraction step of the analysis; liquid extraction, laser ablation, or thermal desorption, and the major component investigated, either the polymer itself or exogenous compounds (additives and contaminants) present within or on the polymer substrate. The review will conclude by summarising some of the challenges these technologies still face and possible directions that would further enhance the utility of ambient ionisation mass spectrometry as a tool for polymer analysis.     

A novel approach of combining thin-layer chromatography with surface-assisted laser desorption/ionization (SALDI) time-of-flight mass spectrometry

A novel means of combining thin-layer chromatography (TLC) with laser desorption/ionization mass spectrometry using a liquid matrix is proposed. Surface-assisted laser desorption/ionization (SALDI) mass spectrometry, which uses a mixture of a micrometer-sized carbon powder (graphite or activated carbon, the SALDI solid) and 15% sucrose/glycerol, dissolved in an equal volume of methanol (SALDI liquid) as a SALDI matrix, is used for laser desorption mass analysis. The ablation of carbon powder from a pencil drawing was used as an alternative to the SALDI solid. The liquid matrix resembled that used in a conventional SALDI matrix system. A line was drawn before separation with a pencil on the track of the sample developed on the TLC plate. After TLC separation, approximately 0.1 microl of SALDI liquid was directly applied to the chromatographic spots on the TLC plate. Porphyrins were used to demonstrate this combination owing to the visible colors of this type of compound. The analyte signal can be easily detected by irradiating the laser along the pencil line on the TLC plate. An additive, p-toluenesulfonic acid, is added to the SALDI liquid to enhance the signal's intensity. This additive dramatically improves the signal-to-noise ratio. A detection limit of approximately 500 pg is demonstrated for porphines, which is 50 times better than that corresponding to conventional TLC SALDI.     

Laser spray: electric field-assisted matrix-assisted laser desorption/ionization

A new liquid chromatography/mass spectrometry interface, the laser spray, has been developed. Explosive vaporization and mist formation occur when an aqueous solution effusing out from the tip of the stainless-steel capillary is irradiated from the opposite side of the capillary by a 10.6 microm infrared laser. Weak ion signals could be detected when the plume was sampled through the ion sampling orifice. When a high voltage (3-4 kV) was applied to the stainless-steel capillary, strong ion signals appeared. The ion abundances were found to be orders of magnitude greater than those obtained by conventional electrospray ionization in the case of aqueous solutions. The present method is regarded as an electric-field assisted form of matrix-assisted laser desorption/ionization in which the liquid chromatographic solvent (water, etc.) acts as a liquid matrix. Laser spray ionization is expected to become a versatile method for biological mass spectrometry because this method is compatible with the natural solvent, water.     

Mass fingerprinting of toxic fractions from the venom of the Indian red scorpion, Mesobuthus tamulus: biotope-specific variation in the expression of venom peptides

The red scorpion, Mesobuthus tamulus, is found in two distinct biotopes within the Indian state of Maharastra-a tropical, sea-level biotope and a semi-arid biotope, up to 600 m. Scorpions from these two geographical areas show marked differences in toxicity. Using mass spectrometry, we have shown biotope-specific variation in the expression of peptides from scorpions collected from these two distinct areas. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) were assessed as techniques for obtaining mass fingerprint data. On line LC/ESI-MS was judged to be the method of choice and unique biotope-specific mass fingerprints, with key diagnostic markers, were obtained.     

A novel approach to analyze membrane proteins by laser mass spectrometry: From protein subunits to the integral complex

A novel laser-based mass spectrometry method termed LILBID (laser-induced liquid bead ion desorption) is applied to analyze large integral membrane protein complexes and their subunits. In this method the ions are IR-laser desorbed from aqueous microdroplets containing the hydrophobic protein complexes solubilized by detergent. The method is highly sensitive, very efficient in sample handling, relatively tolerant to various buffers, and detects the ions in narrow, mainly low-charge state distributions. The crucial experimental parameter determining whether the integral complex or its subunits are observed is the laser intensity: At very low intensity level corresponding to an ultrasoft desorption, the intact complexes, together with few detergent molecules, are transferred into vacuum. Under these conditions the oligomerization state of the complex (i.e., its quaternary structure) may be analyzed. At higher laser intensity, complexes are thermolyzed into subunits, with any residual detergent being stripped off to yield the true mass of the polypeptides. The model complexes studied are derived from the respiratory chain of the soil bacterium Paracoccus denitrificans and include complexes III (cytochrome bc(1) complex) and IV (cytochrome c oxidase). These are well characterized multi-subunit membrane proteins, with the individual hydrophobic subunits being composed of up to 12 transmembrane helices.     

基于微流控芯片-质谱联用的细胞分析研究进展

微流控芯片与质谱联用为细胞研究提供了一个很好的研究平台.质谱的高灵敏度和对化合物独特的鉴别能力可以从复杂的化学信息背景中筛选识别出微量目标物,是细胞分析理想的检测手段.本文重点综述了近年来基于微流控芯片-质谱联用技术的细胞研究进展,从芯片-电喷雾质谱(ESI-MS)接口技术、集成化的样品前处理技术、细胞的药物代谢和细胞相互作用研究及基质辅助激光解吸电离质谱(MALDI-MS)的细胞分析应用等方面总结了最新的方法和技术发展.并展望了芯片-质谱联用新技术应用于细胞分析的可能性.