Parallel SFC/MS-MUX screening to assess enantiomeric purity

Enantiomeric excess (ee) was evaluated for two internally synthesized compound libraries using a high-throughput automated, intelligent four-channel parallel supercritical fluid chromatography/mass spectrometry system equipped with a multiplexed ion source interface (SFC/MS-MUX). The two libraries contained compounds spanning a wide range of enantiomeric ratios with structurally diverse chemical scaffolds and stereogenic centers. The system analyzed each sample simultaneously against four chiral columns using up to six organic modifiers. Enhancements to our previously published parallel supercritical fluid chromatography/mass spectrometry system were implemented to address the challenges associated with automated trace enantiomer identification and quantitation. A reversal of enantiomer elution order was observed for several samples across multiple CSPs and modifiers. The relationship between elution order and % ee accuracy is presented for compounds exhibiting high, middle and low % ee values. Despite incidences in which the minor enantiomer eluted prior to the major enantiomer with less than baseline resolution, the overall % ee was in agreement with separations in which full baseline resolution was achieved. The methods presented here demonstrate the value and utility of high-throughput ee determinations to support drug discovery and development programs.     

Separation of metal chelates and organometallic compounds by SFC and SFE/GC

Supercritical fluid chromatography (SFC) combines the high diffusion coefficients of gas chromatography (GC) and the solubility properties of liquid chromatography (LC). SFC generally requires lower temperatures for chromatographic separations and thus is more suitable for analyzing thermally labile compounds including a number of metal chelates and organometallic compounds. SFC also allows interfacing between supercritical fluid extraction (SFE) and chromatographic analysis of metal-containing compounds. A large number of metal chelates and organometallic compounds can be separated by SFC. This article summarizes SFC separation of various chelates of transition metals, heavy metals, lanthanides and actinides as well as organometallic compounds of lead, mercury, and tin reported in the recent literature. This article also discusses SFC detection systems and the determination of solubility of organometallic compounds by SFC.     

Improved Chiral SFC Screening for Analytical Method Development

In this study we describe the evaluation of a recently developed supercritical fluid chromatography (SFC) instrument for automated chiral SFC method development. The greatly improved gradient dwell volume and liquid flow control of the new instrument in combination with the use of shorter columns containing smaller stationary phase particles affords chiral SFC method development that is faster and more universal than previous systems. Chirality 25:799–804, 2013. © 2013 Wiley Periodicals, Inc.     

Characterization of oligosaccharides from industrial fermentation residues by matrix-assisted laser desorption/ionization,electro spray mass spectrometry,and gas chromatography mass spectrometry

We report here the preliminary characterization of oligosaccharides present in an enzyme-treated industrial fermentation residue using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ion trap mass spectrometry (ESI-ITMS), and gas chromatography mass spectrometry (GC-MS). After sample cleaning with carbon graphite columns, analysis of oligosaccharides present in the sample using MALDI-TOF-MS resulted in identification of molecular ions representing sodiated hexose and pentose oligo/polysaccharides. The GC-MS analyses revealed that the signals observed in the mass spectrum for hexose oligomers represent linear structures, whereas the pentose oligomers were identified as arabinoxylans with a (1-->4) linked Xylp backbone where the Xylp residues were either not substituted or singly substituted with Araf branching residues at positions C-2 or C-3 of the Xylp ring. Analyses by ESI-ITMS of the signals corresponding to arabinoxylan oligosaccharides with four and five monosaccharide residues showed the presence of isomeric structures differing in degree of branching and localization of the branched residue along the Xylp backbone.     

Mapping the primary structure of copper/topaquinone-containing methylamine oxidase fromAspergillus niger

The amino acid sequence of methylamine oxidase (MeAO) from the fungus Aspergillus niger was analyzed using mass spectrometry (MS). First, MeAO was characterized by an accurate molar mass of 72.4 kDa of the monomer measured using MALDI-TOF-MS and by a pI value of 5.8 determined by isoelectric focusing. MALDI-TOF-MS revealed a clear peptide mass fingerprint after tryptic digestion, which did not provide any relevant hit when searched against a nonredundant protein database and was different from that of A. niger amine oxidase AO-I. Tandem mass spectrometry with electrospray ionization coupled to liquid chromatography allowed unambiguous reading of six peptide sequences (11-19 amino acids) and seven sequence tags (4-15 amino acids), which were used for MS BLAST homology searching. MeAO was found to be largely homologous to a hypothetical protein AN7641.2 (EMBL/GenBank protein-accession code EAA61827) from Aspergillus nidulans FGSC A4 with a theoretical molar mass of 76.46 kDa and pI 6.14, which belongs to the superfamily of copper amine oxidases. The protein AN7641.2 is only little homologous to the amine oxidase AO-I (32% identity, 49 % similarity).     

Identification of biotinylated lysine residues in the photoprotein aequorin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mapping after lysine-specific endopeptidase digestion

A method for identifying modified lysine residues in a protein, using lysine-specific endopeptidase treatment followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mapping, is described. As a model protein, the photoprotein aequorin was chosen and the N-hydroxysuccinimide ester of biotin was employed to chemically modify the lysine residues. After digestion with lysine-specific endopeptidase, the biotinylated residues of an amino terminus and five potential lysine residues were identified by MALDI-TOF-MS without any other separation procedure.     

铜绿假单胞菌的MALDI-TOF-MS检测方法的建立

目的 建立利用基质辅助激光解吸电离飞行时间质谱仪( MALDI-TOF-MS)对铜绿假单胞菌的快速检测方法.方法 通过MALDI-TOF-MS法对铜绿假单胞菌进行检测分析,并与生化鉴定方法相比较.结果 MALDI-TOF-MS对铜绿假单胞菌的检测后得到肽指纹图片及相关质谱数据,建立MALDI-TOF-MS对铜绿假单胞菌的快速检测方法.结论 MALDI-TOF-MS方法检测铜绿假单胞菌准确快速、操作简单等特点,可发展成为食品检验铜绿假单胞菌的重要(辅助)工具.     

Applications de la spectrométrie de masse de type MALDI-TOF en microbiologie

In the last few years, matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) has been introduced in clinical laboratories for routine identification. Initially used for research, MALDI-TOF-MS allows rapid and accurate identification of the main microorganisms isolated from clinical samples, by intact cell mass spectrometry or directly from positive blood culture or urines. Other applications are being in processed as bacterial typing, research and detection of antibiotic resistance or particularly virulent strains.     

Solving multicomponent chiral separation challenges using a new SFC tandem column screening tool

A tool for improved tandem column chiral supercritical fluid chromatography (SFC) method development screening was prepared by modification of a commercial analytical SFC instrument with two different software-controllable, six position high-pressure column selection valves, each controlling a bank of five different columns and a pass through line. The resulting instrument, which has the ability to screen 10 different individual columns and 25 different tandem column arrangements, is a useful tool for facilitating the screening of tandem column SFC arrangements for separation of complex mixtures of stereoisomers or other multicomponent mixtures. Strategies for optimal use of the instrument are discussed, and several examples of the use of the instrument in developing tandem SFC methods for resolution of multicomponent mixtures are presented.     

Femtomole oligosaccharide detection using a reducing-end derivative and chemical ionization mass spectrometry

A bimodal reagent (pentafluorobenzyl aminobenzoate) has been synthesized to improve oligosaccharide isolation, detection, and structural characterization. The reagent is glycosidically attached to the reducing end of glycan residues, imparts fluorescent and uv properties for chromatographic detection, and functions as an efficient electron trap under negative ion chemical ionization mass spectrometry for femtomole detectability. Facile ester cleavage and pentafluorobenzyl elimination provides a single molecular-weight-related fragment in high abundance. Procedures are described for reagent synthesis, purification, and oligosaccharide conjugation. Carbohydrate samples derivatized with this reagent are evaluated by high-performance liquid chromatography and supercritical fluid chromatography (SFC) and for sensitivity by SFC negative ion chemical ionization mass spectrometry.     

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.     

A comparison of LC and SFC for cellulose- and amylose-derived chiral stationary phases

This paper presents a systematic comparison of liquid chromatography (LC) and supercritical fluid chromatography (SFC) for Chiralcel OD and Chiralpak AD chiral stationary phases (CSPs), performed using various chiral compounds having a known or potential pharmaceutical activity. The chiral recognition mechanisms involved in LC and SFC for the enantiomeric separation of β-blockers have been studied more particularly. As a general rule, it appears that the presence of polar functions, like primary or secondary hydroxyl or amine functions, may result in marked discrepancies in selectivity between LC and SFC. This result is peculiar to cellulose- and amylose-derived CSPs, for which the interactions involved in chiral recognition mechanism are not always well balanced, contrary to what happens for independent CSPs. In the case of chiral resolution of polar solutes or polymer-type CSPs, the analyst should try both the LC and SFC techniques to be able to choose the more stereoselective one. © 1995 Wiley-Liss, Inc.     

Tuning of mobile and stationary phase polarity for the separation of polar compounds by SFC

The separation of polar compounds by supercritical-fluid chromatography (SFC) is reviewed. New developments in mobile and stationary phase tuning are reviewed for packed and packed capillary SFC. In terms of mobile phase polarity adjustment, new pure and multiple component fluids are presented. The approach of tuning the polarity of the stationary phase as a way of increasing the range of polar compounds analyzed by SFC using pure CO(2) is discussed using either silica-based or new materials as stationary phase. Chiral, liquid crystal and polymer-based stationary phases coated on particles are widely covered in this review as an interesting approach to separate polar compounds avoiding the major drawbacks associated to the use of modifiers in SFC.     

生物质谱技术在糖蛋白结构分析中的应用

生物质谱包括基质辅助激光解吸附飞行时间质谱及电喷雾质谱被广泛应用于生物样品如多肽、蛋白质及核酸的分析,由于这种具有软电离方式的质谱具有极高的灵敏度及准确度,目前也被成功地用于糖蛋白的结构分析,与普通的化学方法相比,质谱法快速、简单,结合网上数据库检索、凝集素亲和提取、二维凝胶电泳以及靶上直接酶切等新方法,可以提供糖蛋白的一级结构乃至高级结构的信息。     

微生物学研究中的软电离质谱技术

包括基质辅助激光解吸电离（ＭＡＬＤＩ）和电喷雾（ＥＳＩ）在内的软电离质谱是最近发展起来的质谱技术,由于这些电离方式对样品的破坏性小,质量测定范围大,分子量测定准确,样品纯度要求不高很适合分析成分复杂的微生物样品,ＭＡＬＩＤＩ－ＴＯＦ－ＭＳ结合高分辨率的二维ＳＤＳ－ＰＡＧＥ可以分析１０＾－１２摩尔水平的蛋白,是细菌蛋白质研究过程中必不可少的工具。最近的研究工作表明,通过ＭＡＩＤＩ－ＴＯＦ－ＭＳ或ＨＰ     

Analysis of hydrolytic activity of phospholipase Calpha from porcine retina on retinyl ester and phosphatidylcholine using non-denaturing two-dimensional electrophoresis and mass spectrometry

Hydrolysis of retinyl esters and phospholipids is important for visual functions of the animal retina. This study aimed to examine hydrolytic activity of an enzyme with native substrates such as retinyl esters and phospholipids responsible for this function in porcine retina. After cytosolic proteins were extracted from porcine retina, the proteins were separated using non-denaturing two-dimensional electrophoresis (2DE). Some major proteins and phospholipase Calpha were identified by matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) or electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). The phospholipase Calpha showed hydrolytic activities with not only alpha-naphtyl acetate but also with retinyl palmitate and phosphatidylcholine when effects of different substrates were investigated using enzyme activity staining on 2DE or MALDI-TOF-MS. Results indicated that hydrolytic activity of the enzyme with non-native and native substrates could be examined using a combination of non-denaturing 2DE and MALDI-TOF-MS.     

Interactions of arsenic with human metallothionein-2

Arsenic is a toxic element that is found in the atmosphere, as well as in aquatic and terrestrial environments. We have demonstrated that As(3+) binds to human metallothionein-2 (hMT-2) by UV absorption spectroscopy, inductively coupled plasma-atomic emission spectrometry (ICP-AES), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MALDI-TOF-MS revealed that the structure of the adduct formed by arsenic and hMT-2 (As-hMT-2) was not homogeneous. The maximum molar ratio of arsenic to hMT-2 was found to be more than 6:1 on ICP-AES, UV absorption spectroscopy and MALDI-TOF-MS. The ratio of the number of sulfhydryl groups in hMT-2 that bound arsenic was 3:1, which is the same as the ratios reported previously for arsenic-glutathione and arsenic-phytochelatin complexes.     

Enantiomer separation by complexation SFC on immobilized Chirasil-nickel and Chirasil-zinc

The use of complexation SFC for enantiomer separation of Lewis base selectands on chiral nickel(II)- and zinc(II)-bis[(3-heptafluorobutanoyl)-10-methylene-(1R)-camphora te] chemically bonded to poly(dimethylsiloxane) (Chirasil-nickel and Chirasil-zinc) and employed as Lewis acid selectors is described. The method is especially suited for less volatile and configurationally labile racemates. The variation of the experimental parameters temperature T, pressure p and density rho of the mobile phase carbon dioxide on the retention factor k, relative retention r and chiral separation factor alpha is studied, providing insights into the mechanisms of chiral recognition under supercritical conditions. For mecoprop methyl ester (methyl 2-(4-chloro-2-methylphenoxy)propanoate) an unusual increase of alpha at increased temperature is observed on Chirasil-nickel. Supercritical carbon dioxide does not inadvertently affect the complexation equilibria between Lewis donor selectands and the Lewis acid metal selectors during complexation SFC.     

Proteomic technologies for identification of serum potential biomarkers of autoimmune demyelinating polyneuropathies

Time-of-flight MALDI mass spectrometry (MALDI-TOF-MS) profiling of blood serum of patients with Guillain-Barré syndrome (GBS, 36 samples), chronic inflammatory demyelinating polyneuropathy (CIDP, 24 samples) and practically healthy donors (HD) (35 samples) was carried out in order to identify potential biomarkers of autoimmune demyelinating polyneuropathies (ADP). To simplify the peptide-protein mixture of serum prior to MALDI-TOF-MS analysis samples were pre-fractionated on magnetic microparticles with a weak cation-exchange (MB-WCX) surface. Comparative analysis of mass spectrometric data using the classification algorithms (genetic and neural network-controlled) revealed a characteristic set of peaks, agreed change area with a high specificity and sensitivity of the differentiated mass spectrometry profiles of the blood serum of patients with DPNP and healthy donors (for GBS values of these characteristics reached 100 and 100, and for CIDP 94.1 and 100% respectively). Comparative analysis of mass spectrometric profiles of serum samples obtained from patients with GBS and CIDP, allowed to build a classification model to differentiate these diseases from each other, with a specificity of 88.9 and a sensitivity of 80%.     

Evaluation of sphingolipids in vitreous bodies from a patient with Gaucher disease, using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Gaucher disease is a glycolipid storage disorder characterized by the accumulation of glucosylceramide in tissues. Using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry (DE MALDI-TOF-MS), we analyzed sphingolipids in vitreous bodies from a patient with Gaucher disease who suffered from vitreous opacities. Crude lipids were extracted from the freeze-dried vitreous bodies with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by DE MALDI-TOF-MS. The results were as follows: (a). the m/z values of the ions found in the mass spectra for both the control and the Gaucher disease patient corresponded to different sphingomyelin species. (b). The mass spectrum of the Gaucher disease patient showed additional ions with m/z values corresponding to different ceramide monohexoside (CMH) species. It was indicated that the accumulation of CMH in vitreous bodies from Gaucher disease patients could be easily detected with the DE MALDI-TOF-MS method.