A comprehensive characterization of the T-cell antigen receptor complex composition by microcapillary liquid chromatography-tandem mass spectrometry

It has become apparent that many intracellular signaling processes involve the dynamic reorganization of cellular proteins into complex signaling assemblies that have a specific subunit composition, function, and subcellular location. Since the elements of such assemblies interact physically, multiprotein signaling complexes can be isolated and analyzed. Recent technical advances in highly sensitive protein identification by electrospray-tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed. The T-cell antigen receptor (TCR) is an oligomeric transmembrane protein complex that is essential to T-cell recognition and function. The extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor-ligand interactions. We used microbore capillary chromatography-tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cells of the murine T-cell line CD11.3. We identified all the previously known subunits of the TCR/CD3 complex as well as proteins previously not known to associate with the TCR. The catalytic activities of some of these proteins could potentially be used to interfere pharmacologically with TCR signaling.     

基于整体材料的微流控芯片反相液相色谱-串联质谱平台用于蛋白质分析

微流控芯片高效液相色谱-串联质谱系统具有高通量、高灵敏度等优点,已成为生物样品分析的热点领域之一。本文在玻璃芯片上以甲基丙烯酸十二酯(LMA)和三羟甲基丙烷三甲基丙烯酸酯(TMPTMA)为单体,制备了以聚丙烯酸酯整体材料为固定相的捕集柱和分离柱。通过在芯片通道末端连接细内径的毛细管作为芯片-质谱接口,并以常规的液相色谱泵和微阀控制流体,构建了芯片反相液相色谱-电喷雾串联质谱(RPLC-ESI-MS/MS)平台,并将其用于分析牛血清白蛋白(BSA)的酶解产物。经过3次平行分析,BSA的序列覆盖率分别为39.37%、37.89%和34.10%(相对标准偏差为7.3%)。采用不同批次制作的芯片构建RPLC-ESI-MS/MS平台,对BSA酶解产物进行分析,其序列覆盖率相当。上述结果表明,该平台具有灵敏度高和重现性好等优点,有望用于蛋白质样品的快速分离和高灵敏度鉴定。     

Development of a method for the identification of azaspiracid in shellfish by liquid chromatography-tandem mass spectrometry

Azaspiracid is the main toxin responsible for a number of recent human intoxications in Europe resulting from shellfish consumption. The first micro liquid chromatography-tandem mass spectrometry (micro-LC-MS-MS) method was developed for the determination of this novel shellfish poisoning toxin in mussels. The analyte was extracted from whole mussel meat with acetone and chromatographed on a C18 reversed-phase column (1.0 mm I.D.) by isocratic elution at 30 microl/min with acetonitrile-water (85:15, v/v), containing 0.03% trifluoroacetic acid. The toxin was ionised in an ionspray interface operating in the positive ion mode, where only the intact protonated molecule, [M+H]+, was generated at m/z 842. This served as precursor ion for collision-induced dissociation and three product ions, [M+H-nH2O]- with n=1-3, were identified for the unambiguous toxin confirmation by selected reaction monitoring LC-MS-MS analysis. A detection limit of 20 pg, based on a 3:1 signal-to-noise ratio, was achieved for the analyte. This LC-MS-MS method was successfully applied to determine azaspiracid in toxic cultivated shellfish from two regions of Ireland.     

高效液相色谱-串联质谱法分离鉴定单唾液酸四己糖神经节苷脂中的杂质

用高效液相色谱法（HPLC）分析了单唾液酸四己糖神经节苷脂(GM1)药物的冻干粉、中间体和注射液各6个批号的样品,以期查找出采用不同工艺生产的成品中造成临床副反应的可能杂质。在用HPLC对多个批号GM1成药的对比分析中,发现某个批号的GM1干粉中多出2个杂质峰,其保留时间与制作该批号的GM1中间体的2个杂质峰保留值相同。对该批号的GM1干粉和相应批号的GM1中间体中的目标杂质进行半制备级收集,经冷冻干燥除溶剂富集35倍后,用电喷雾-四极杆-飞行时间质谱（ESI-Q-TOF MS）定性分析,对主分子离子峰进行了二级质谱分析。经谱图解析后,推断在GM1干粉和中间体中存在的保留值相同的杂质可能是同种组分。两个杂质峰的结构皆为岩藻糖(Fuc)-GM1,仅是神经酰胺上的长链基团有所不同,峰1的该基团由16个CH2组成,而峰2的该基团由18个CH2组成。临床数据表明含有上述两种杂质的2批GM1注射液都有副反应,而没有上述杂质的4批GM1注射液都没有临床副反应,而且这两批有临床副反应的GM1注射液都是用同一个批号的含有这两种杂质的中间体制成的,因此推断上述两个杂质可能是引起副反应的主要组分。     

Method development for identification of ketobemidone metabolites in microdialysate samples by coupled-column capillary liquid chromatography-tandem mass spectrometry

Methodologies for identification of ketobemidone metabolites in microdialysate samples utilizing coupled-column capillary liquid chromatography-electrospray quadrupole time-of-flight tandem mass spectrometry are presented. Two different methods were developed to efficiently analyze the metabolites norketobemidone, ketobemidone N-oxide and hydroxyketobemidone, respectively. Both methods include on-line desalting and trapping of the analytes on micro-solid-phase extraction columns with different retention mechanisms. Norketobemidone and ketobemidone N-oxide were trapped on a C18 column and then eluted by back-flush followed by isocratic separation on a fluorinated reversed-phase type silica gel column (Fluofix). Retention equations are proposed for the chromatographic observations made on the Fluofix column. Hydroxyketobemidone was trapped on a phenylboronic acid column by complex formation at basic pH and then eluted at acidic pH directly into to the mass spectrometer. Oxidation of hydroxyketobemidone to its corresponding quinone was also observed. The methods were successfully used to analyze synthetic ketobemidone metabolites in dilute low-volume microdialysis samples.     

Characterization of C-glycosyl flavones O-glycosylated by liquid chromatography-tandem mass spectrometry

Fifty three O-glycosyl-C-glycosyl flavones with O-glycosylation on phenolic hydroxyl or on the C-glycosyl residue or combination of both forms have been studied by liquid chromatography-UV diode array detection-electrospray ionisation mass spectrometry ion trap in the negative mode. The study of the relative abundance of the main ions from the MS preferential fragmentation on -MS2 and/or -MS3 events allows the differentiation of the position of the O-glycosylation, either on phenolic hydroxyl or on the sugar moiety of C-glycosylation. In addition, it is possible to discriminate between O-glycosylation at 2' and at 6' positions. The occurrence of an abundant ion Y(0)(-) ([(M-H)-132/-146/-162](-), mono-O-pentosyl/rhamnosyl/hexosyl-C-glycosyl derivatives) after -MS2 fragmentation characterizes the O-glycosylation on phenolic hydroxyls. The preferential fragmentation leading to a relevant Z(1)(-) ([Y(1)-18](-)) fragment is characteristic of 2'-O-glycosyl-C-glycosyl derivatives. The 6'-O-glycosyl-C-glycosyl derivatives are characterized by (0,2)X(0)(-), which is generated by a global loss of the sugar moiety from the O-glycosylation at 6' and the glycosidic fraction that involves the carbons 6'-3' of the C-glycosyl residue ([(M-H)-162-120](-), in the case of 6'-O-hexosyl-C-hexosyl derivatives). Regarding the combined O-glycosylated compounds (both on phenolic hydroxyl and on sugar moiety at C-glycosylation), the main fragmentation on -MS2 events produces a Y(0)(-) characterizing the O-glycosylation on the phenolic hydroxyl, and the -MS3[(M-H)-->Y(0)](-) fragmentation of the O-glycosylation on the C-glycosyl residue.     

液相色谱-串联质谱法测定水环境中的十氯酮

建立了分析测定水环境中十氯酮的液相色谱-串联质谱法。水样经液液萃取、净化后,采用Eclipse plus C18柱（100 mm×2.1 mm,3.5 μm）分离,乙腈和水为流动相进行梯度洗脱,在电喷雾负离子多反应监测模式下进行检测,同位素内标法定量。结果表明：采用液相色谱-质谱联用技术,证实了十氯酮在甲醇中以半缩醛的形式存在,而在丙酮/乙腈中以偕二醇的形式存在。由于十氯酮极性较强,在净化时难以洗脱,并且不耐酸,所以不能与其他有机氯农药一起分析。十氯酮在5~100 μg/L范围有良好的线性关系,相关系数r²=0.999,检出限及定量限分别为0.70 ng/L和2.8 ng/L;在5、40和100 ng/L 3个浓度添加水平的平均回收率为95.1%~98.9%,相对标准偏差为3.85%~4.72%。本方法具有良好的灵敏度、回收率和重现性,适用于水环境中十氯酮的测定。     

High-throughput multiclass method for antibiotic residue analysis by liquid chromatography-tandem mass spectrometry

A simple and rapid method has been developed for the residue analysis of 39 antibiotics (tetracyclines, quinolones, penicillins, sulfonamides and macrolides) in foodstuffs of animal origin. The method combines an effective extraction technique, which uses water-methanol as extracting solvent, with ultra-high-pressure liquid chromatography-tandem mass spectrometry, allowing both confirmation and quantification in a single chromatographic run. The multiresidue method has been validated in chicken muscle matrix according to European Union Decision 2002/657/EC. It has been implemented as a routine method in a Public Health Laboratory, instead of the five plates test and LC methods previously used.     

A liquid chromatography-tandem mass spectrometry method for fluazifop residue analysis in crops

An LC-MS-MS assay is described for fluazifop residue analysis in crops. The residues are extracted with acidified organic solvent, the esters and conjugates are hydrolysed with 6 M hydrochloric acid, then the extracts are cleaned-up by solid phase extraction using C2(EC) and Si cartridges in tandem. Quantitative analysis is performed by gradient liquid chromatography coupled to triple quadrupole mass spectrometer using atmospheric pressure chemical ionisation. All fluazifop-P-butyl, free fluazifop-P and any conjugates are quantified as fluazifop-P. The limit of quantification is 0.01-0.05 mg/kg depending on crop matrices. The clean-up method is also suitable for LC-UV analysis with a compromise in higher limit of quantification 0.05-0.2 mg/kg.     

A liquid chromatography-tandem mass spectrometry method for fluazifop residue analysis in crops

An LC-MS-MS assay is described for fluazifop residue analysis in crops. The residues are extracted with acidified organic solvent, the esters and conjugates are hydrolysed with 6 M hydrochloric acid, then the extracts are cleaned-up by solid phase extraction using C₂(EC) and Si cartridges in tandem. Quantitative analysis is performed by gradient liquid chromatography coupled to triple quadrupole mass spectrometer using atmospheric pressure chemical ionisation. All fluazifop-P-butyl, free fluazifop-P and any conjugates are quantified as fluazifop-P. The limit of quantification is 0.01–0.05 mg/kg depending on crop matrices. The clean-up method is also suitable for LC-UV analysis with a compromise in higher limit of quantification 0.05–0.2 mg/kg.     

Tryptic peptide analysis of protein binders in works of art by liquid chromatography-tandem mass spectrometry

A proteomics approach was used for the identification of protein binders in historical paints: the proteins were digested enzymatically into peptides using trypsin before being separated and detected by high performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS). Mascot (Matrix Science) was used to analyse the resulting data and for protein identification. In contrast to amino acid analysis, amino acid sequences could be studied that retain much more information about the proteins. The best extraction strategy was selected based on the number of peptides that were identified in the protein content of paint replicas using different methods. The influence of pigments on the extraction method was studied and the analytical characteristics of the selected method were determined. Finally this method was applied to historical paint microsamples on the anonymous early 15th century panel painting Crucifixion with St Catherine and St Barbara (Calvary of the Tanners), the St Catherine Altarpiece by Joes Beyaert (c. 1479) and two paintings by Pieter Brueghel the Younger (1617-1628).     

Applicability of ultra-performance liquid chromatography-tandem mass spectrometry for heroin profiling

The applicability of ultra- performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) for heroin profiling is described. The coupling of the high separation power of UPLC with the highly selective and sensitive detection of MS/MS is well suited for heroin profiling. An Acquity UPLC BEH C18 1.7 microm particle column (100 mm x 2.1mm) with binary gradients containing 1% formic acid (pH 2.0) or 10 mM ammonium bicarbonate (pH 10.0)/acetonitrile mixtures was investigated for the profiling. For MS/MS detection, an atmospheric pressure positive electrospray source was employed with multiple reaction monitoring (MRM). MRMs for individual basic impurities were generated for heroin profiling using low and high pH mobile phases, while MRMs for neutral impurities were generated using a high pH mobile phase. Compared to a pH 2.2 mobile phase, the use of a pH 10 mobile phase allowed for significantly greater sample loading, major selectivity differences, and lower MRM sensitivity. UPLC-MS/MS allowed for the highly selective and sensitive detection of many of the targeted solutes in seized heroin exhibits. Basic impurities detected included morphine, codeine, noscapine, papaverine and the previously unreported solutes reticuline, reticuline monoacetate (2 products), reticuline diacetate, narceine, codamine, laudanidine, cryptopine, laudanosine, and norlaudanosine. Neutral impurities found included N,3,6-triacetylnormorphine, N-acetylnorcodeine, N-acetylnornarcotine, 3,6-dimethoxy-4-acetyloxy-5-[2-(N-methylacetamido)]-ethylphenanthrene, and cis-n-acetylanhydronornarceine. The detection of these impurities, at levels as low as 10(-6)% w/w should allow for greatly enhanced heroin profiles.     

Hardware-accelerated protein identification for mass spectrometry

An ongoing issue in mass spectrometry is the time it takes to search DNA sequences with MS/MS peptide fragments (see, e.g., Choudary et al., Proteomics 2001; 1: 651-667.) Search times are far longer than spectra acquisition time, and parallelization of search software on clusters requires doubling the size of a conventional computing cluster to cut the search time in half. Field programmable gate arrays (FPGAs) are used to create hardware-accelerated algorithms that reduce operating costs and improve search speed compared to large clusters. We present a novel hardware design that takes full spectra and computes 6-frame translation word searches on DNA databases at a rate of approximately 3 billion base pairs per second, with queries of up to 10 amino acids in length and arbitrary wildcard positions. Hardware post-processing identifies in silico tryptic peptides and scores them using a variety of techniques including mass frequency expected values. With faster FPGAs protein identifications from the human genome can be achieved in less than a second, and this makes it an ideal solution for a number of proteome-scale applications.     

Characterization and identification of pradimicin analogs from Actinomadura hibisca using liquid chromatography-tandem mass spectrometry

Microbial cultures produce complex and potentially interesting mixtures of biosynthetic intermediates and derivatives of metabolites. These mixtures' reliable identification is important and so too is the development of techniques for their analysis. Here, a simple and highly selective method of detecting the biosynthetic congeners involved in the pentangular polyphenol pradimicin (PR) pathway from Actinomadura hibisca fermentation was developed. Solid-phase extraction (SPE) cleanup using an OASIS HLB cartridge was a simple and reliable tool for the extraction of PRs from a fermentation broth. The separation of each natural PR analog--eluted with a gradient system of aqueous acetonitrile through a reversed-phase C(18) column containing ammonium acetate and acetic acid as additives--allowed their simultaneous profiling. The combined use of SPE cleanup and chromatographic separation, coupled with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) detection was demonstrated to be sufficiently accurate and reliable to analyze the natural PR analogs produced from A. hibisca. Ten natural PRs were identified: four alanine-containing (PRA, PRC, PRL, and PRB), two glycine-substituted (PRD and PRE), and four serine-substituted (PRFA-1, PRFA-2, PRFL, and PRFB). This report demonstrates the first use of both SPE cleanup and HPLC-ESI-MS/MS to profile a wide range of structurally closely related PRs in a bacterial fermentation broth.     

One-step functionalization of paper and simplified antibody immobilization for on-the-spot immunocapture from dried serum in liquid chromatography-tandem mass spectrometry based targeted protein determination

This work aimed to simplify and improve the process of binding monoclonal antibodies (mAbs) covalently to filter paper for use in dried blood spot sampling, enabling instant capture of protein biomarkers for targeted protein determination. Incorporating the necessary immunocapture sample preparation step in the initial sampling stage saves time and reduces the workload. The biomarker human chorionic gonadotropin (hCG) was used as the model analyte. The antibody-based paper samplers were prepared by functionalizing paper discs (6 mm) through a simple reaction using divinyl sulfone (DVS). After DVS activation, the paper discs were incubated with E27 hCG mAbs, followed by 0.05% tween/phosphate buffer saline to block the surface. After sample application and drying, the discs only needed to be washed before tryptic digestion and finally analysed on a nanoliquid chromatography–tandem mass spectrometry system. The finished DVS-mAbs samplers could selectively capture hCG (100 ng/mL) from human serum, with a recovery of 50%. Sample clean-up reduced the number of identified proteins from 132 to 82 before and after wash, respectively, with a 70% reduction in serum albumin signal while still retaining hCG on the sampler during the washing protocol. An evaluation of the samplers revealed excellent linearity (R² = 0.9995) for hCG in serum with relative standard deviations below 15%. This work has presented the first ever reported paper samplers immobilized with antibodies utilizing DVS chemistry, showing promise in the future of paper-based sampling.     

Comprehensive characterization of rodenticides in wastewater by liquid chromatography-tandem mass spectrometry

Rodenticides are used as pest control to eradicate rodents and have emerged as new environmental contaminants due to their widespread use in domestic and urban infrastructures. In this study, we have developed and validated an analytical methodology based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of 13 anticoagulant rodenticides in wastewater. In a first step, ionization conditions were tested in electrospray mode, and positive ionization gave the highest sensitivity. Fragmentation patterns were determined and two selected reaction monitoring (SRM) transitions were selected for each compound. Using a Zorbax Eclipse XDB-C18 column and specific SRM transitions, 13 compounds were resolved. The LC-MS/MS method provided good linearity, sensitivity, intra- and inter-day precision, and good identification capabilities for these compounds in wastewaters. Thereafter miniaturized liquid–liquid extraction (LLE) and solid-phase extraction (SPE) were optimized. Oasis HLB and Strata WA SPE cartridges with methanol/dichloromethane as eluting solvents provided good recoveries and limits of detection ranged between 0.34 and 20 ng L^?1, whereas LLE failed to recover some compounds. Finally, the performance of both LLE and SPE methods was evaluated by analyzing rodenticides in a set of wastewaters. Warfarin was the only detected compound at nanogram per liter level, and good agreement was observed between LLE and SPE.     

基于液相色谱-串联质谱的结肠癌血清氧固醇标志物筛选

结肠癌(CC)是全球常见恶性肿瘤之一,发病率呈逐年上升趋势,目前没有有效的标志物用于疾病早期诊断和干预跟踪。胆固醇及其氧化衍生物氧固醇在众多恶性肿瘤发生发展中发挥关键作用。该研究采用液相色谱-串联质谱(LC-MS/MS)技术,对CC临床血清样本中胆固醇及相关10种氧固醇代谢物进行了定性定量分析,并采用偏最小二乘判别分析(PLS-DA)和正交偏最小二乘判别分析(OPLS-DA)进行多元统计分析,发现上述目标代谢物能够较好地区分CC组与健康对照组。为防止数据过拟合,该研究在PLS-DA模型各代谢物变量投影重要性(VIP)基础上,结合最优组分数及K-均值聚类结果,筛选得到3种代谢标志物。通过受试者操作特征曲线(ROC)的曲线下面积(AUC)分析,发现筛选得到的3种潜在标志物联合预测CC达到0.998,说明模型性能优良。GO(基因本体论)富集分析显示3种潜在标志物主要分布在内质网和包被囊泡上,参与胆固醇代谢、运输、低密度脂蛋白重塑等生物进程,发挥胆固醇运输活性和低密度脂蛋白颗粒受体结合的分子功能。KEGG(京都基因与基因组百科全书)通路分析显示3种潜在标志物富集于类固醇生物合成、PPAR(过氧化物酶体增殖物激活受体)信号通路及ABC(ATP结合盒)转运等通路上。该研究为寻找CC标志物及进一步阐明胆固醇及氧固醇在CC发病过程中的作用奠定了一定的基础。     

液相色谱-串联质谱法同时测定化妆品中的25种喹诺酮类药物

建立了直接提取结合液相色谱-电喷雾串联质谱同时测定化妆品中丹诺沙星、恩诺沙星、氟甲喹、恶喹酸、环丙沙星、沙拉沙星、萘啶酸、诺氟沙星、氧氟沙星等25种喹诺酮类药物的方法。样品经酸性乙腈提取和正己烷脱脂净化,采用Poroshell EC-C₁₈色谱柱分离,含0.1%甲酸的乙腈-水溶液为流动相进行梯度洗脱,电喷雾串联质谱正离子模式扫描,多反应监测(MRM)模式检测,外标法定量。实验通过空白基质液配制标准溶液,以降低基质对离子化干扰造成的基质效应,25种喹诺酮类药物在1~200 mg/kg范围内线性关系良好,相关系数(r)在0.999以上。方法的检出限为1.0 mg/kg,在1、5、10 mg/kg 3个加标水平下,水、乳、霜型化妆品中加标回收率为87.4%~105%,相对标准偏差(RSD)为4.54%~19.7%(n=6)。结果表明,该方法简便、快速、准确,适用于化妆品中25种喹诺酮类药物的测定。     

Analysis of p-chlorobenzoic acid in water by liquid chromatography-tandem mass spectrometry

para-Chlorobenzoic acid (p-CBA) is typically used as a probe compound to indirectly quantify hydroxyl radicals formed during advanced oxidation processes used in drinking water and wastewater treatment. A method has been developed for the sensitive analysis of p-CBA in water using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A reporting limit in water of 100 ng/L was determined for the method, which is 40-fold lower than the 4.0 microg/L reporting limit of the widely used liquid chromatography with UV detection (LC-UV) method. The method was found to be robust in difficult matrices such as wastewater and highly selective, unlike LC-UV which relies on non-specific detection at 234 nm. The detection of p-CBA below 1 microg/L during bench-scale ozonation of wastewater after hydrogen peroxide addition was demonstrated. Duplicate samples were analyzed by LC-MS/MS and LC-UV and results were found to be comparable at concentrations quantifiable by both methods.     

高效液相色谱-串联质谱法检测三苯乙烯基苯酚聚氧乙烯醚

建立了高效液相色谱-串联质谱检测三苯乙烯基苯酚聚氧乙烯醚(tristyrylphenol ethoxylates,TSPn EO)的分析方法,并对TSPn EO在反相色谱柱(XBridge C_(18),150 mm×2.1 mm,3.5μm)、亲水相互作用色谱柱(XBridge HILIC,150 m m×2.1 m m,3.5μm)、氨基色谱柱(XBridge Amide,150 m m×2.1 m m,3.5μm)、伪反相色谱柱(C18柱(XBridge C_(18),50 mm×2.1 mm,5μm)与硅胶柱(Nova-Pak Silica,150 mm×2.1 mm,4μm)串联)4种不同液相色谱分离模式下的分离效果进行了研究。实验比较了5 mmol/L乙酸铵水-乙腈、0.1%(v/v)甲酸水-乙腈、水-乙腈和水-甲醇4种流动相组成及3种梯度洗脱条件对分离效果和灵敏度的影响。探讨了TSPnEO在电喷雾离子(ESI)源内的离子化特征,结果表明,在ESI正离子模式下,TSPnEO在离子源内形成[M+NH4]+离子,其聚合度的分布特征符合泊松分布。利用伪反相色谱柱,水-乙腈作为流动相,实现了不同聚合度(n=5~18)TSPnEO的分离。