基于LTQ-Orbitrap液相色谱-质谱联用技术的乳源乳清蛋白组分的差异性研究

利用LTQ Orbitrap XL组合型傅立叶变换高分辨质谱系统分析了乳源蛋白主要组分肽指纹图谱。对南方水牛乳与不同来源的乳清蛋白的氨基酸序列研究结果表明,乳清蛋白经酶解后主要为α-乳白蛋白(α-La)和β-乳球蛋白(β-Lg)组分,乳清蛋白肽质指纹谱的分析显示水牛乳与荷斯坦奶乳清蛋白α-La氨基酸发生变异的比率明显少于山羊奶乳清蛋白α-La,说明荷斯坦奶α-La和水牛乳α-La的差异更小,同源性更强;而水牛乳β-Lg与荷斯坦乳β-Lg氨基酸发生变异的部位比率要多于山羊奶,水牛乳β-Lg与山羊奶同源性更强;乳源酪蛋白酶解后的肽段主要组分为αs1-CN,β-CN,κ-N,通过对水牛乳酪蛋白的氨基酸序列的差异性分析,不同品种的乳源酪蛋白的氨基酸序列明显存在差异。与乳清蛋白相比,奶牛品种差异导致乳蛋白发生氨基酸差异现象更显著,酪蛋白的氨基酸序列对比表明,水牛奶酪蛋白与山羊奶酪蛋白比与乳牛酪蛋白的差异更大。     

牛奶中酞酸酯污染物的气相色谱质谱-联用测定

建立了气相色谱.质谱法(GC-MS)测定市售牛奶中4种酞酸酯(邻苯二甲酸二乙酯(DEP)、邻苯二甲酸二异丁基酯(DiBP)、邻苯二甲酸二丁基酯(DBP),邻苯二甲酸二(2-乙基己基)酯(DEHP))的方法.样品采用乙酸乙酯直接萃取,C18小柱净化,过0.45μm滤膜,直接注入GC-Ms进行分析.保留时间定性,外标法定量.4种酞酸酯的回收率为77.9%～109.07%;精密度(RSD)为1.72%～6.18%;检测限在0.15～60 ng之间.     

乙酰化蛋白的质谱鉴定研究

报道了两种生物质谱技术ESI-MS和MALDI-MS在鉴定乙酰化修饰蛋白BSA-ac中的应用研究结果. 乙酰化修饰蛋白通过特征碎裂峰m/z 126.1或MS/MS质谱图中相差一个赖氨酸的相邻b或y离子之间170 Da分子量的差异确证赖氨酸乙酰化修饰, 并且后者提供具体修饰位点信息. 研究提示ESI-MS和MALDI-MS两种质谱技术均可用于鉴定实际复杂样品中的乙酰化蛋白, 且在乙酰化蛋白的鉴定中各有其优点.     

毛细管电泳质谱联用技术及其应用

本文介绍了用于毛细管电泳质谱联用仪器的多种接口技术，描述了ＣＺＥ，ＣＩＥＦ，ＣＧＥ，ＭＥＫＣ和ＣＩＴＰ等毛细管电泳技术和四极质谱，离子阱质谱，傅 叶变换离子回旋共振质谱，飞行时间质谱，磁质谱，解吸质谱等联用的现状及发展前景，对近年来ＣＥ－ＭＳ在酶解产物。蛋白质和肽，核苷酸，药物及代谢产物等领域中的应用作了详细述评。     

气相色谱-质谱联用技术测定水中油

本文综述了近年来用气相色谱法测定水中油的技术进展。引用文献13篇。     

现代质谱及其联用技术在抗癌药物研究中的应用

综述了质谱及其联用技术(如气相色谱-质谱,液相色谱-质谱,毛细管电泳-质谱等)在抗癌药物的分离鉴定、药物代谢产物的研究及药物作用机理方面的应用,总结了其发展潜力。     

质谱联用技术在生物大分子分析中的应用

本文评述了近年来质谱联用技术在生物大分子分析应用中的最新进展。着重评述了由于ESI接口的出现而推动的高效液相色谱一连续流动快速原子轰击质谱(HPLC-cfFABMS )、高效取代色谱一连续流动快速原子轰击质谱(HPDC-cfFABMS ) ,凝胶渗透色谱一诱导祸合等离子体质谱(GPC-1CPMS )、毛细管电泳一质谱(CE-MS )和毛细管电泳一飞行时间质谱(CE-TOFMS)等方面近两年的动向及成果。本文较广泛地展示了质谱联用技术在生物大分子分析中的重要作用和独特的地位。     

液相等电聚焦技术结合液相色谱-质谱联用技术分析鼠肝全蛋白

利用液相等电聚焦预分离技术结合液相色谱-质谱(LTQ-Orbitrap)联用技术,研究了C57小鼠肝脏的蛋白质表达谱.质谱分析结果采用Max Quant1.4.1.2软件搜索数据库,共鉴定出3474个蛋白(2个以上唯一肽段).用DAVID在线工具、GO分类工具和IPA软件对鉴定蛋白进行生物信息学分析,单独发现832个新蛋白.研究结果表明,通过基于等电点和亲疏水性的两维分离,提高了质谱鉴定蛋白数,可以鉴定出更多低丰度蛋白.     

金属硫蛋白的色谱/电喷雾电离质谱联用表征方法

通过反相液相色谱(RPLC)与电喷雾电离质谱(ESI-MS)的联用技术,对镉诱导金属硫蛋白标准物质MT-1和MT-2的结构进行表征分析。采用Vydac C8 反相色谱柱(250 mm×2.1 mm i.d., 5 μm, 30 nm),流动相A为pH 6.0的5 mmol/L乙酸铵水溶液,流动相B为pH 6.0的5 mmol/L乙酸铵的甲醇-水(体积比为1∶1)溶液,流动相流速为0.20 mL/min,在40 min内流动相B的体积分数从10%增加到37.5%进行梯度洗脱。分别用紫外(UV)和ESI-M     

高效液相色谱-质谱联用技术的应用进展

高效液相色谱-质谱联用技术具有高分离能力、高灵敏度、应用范围广和极强的专属性等特点。对高效液相色谱-质谱联用技术在药物分析、食品分析和环境分析等领域的应用,特别是在中草药成分分析、中药指纹图谱研究、药物代谢研究、体内药代动力学研究、西药及中成药成分分析、药物筛选研究等方面的应用进行了综述。     

Casein phosphoproteome: identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresis

We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five beta-, fifteen alpha(s1)-, ten alpha(s2)-, and four kappa-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to kappa-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single kappa-CN component. The phosphate group on site Ser12 of tryptic peptide 8-22 of most phosphorylated alpha(s1)-CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two kappa-CN components was determined by means of MS/MS analysis.     

Analysis of major ovine milk proteins by reversed-phase high-performance liquid chromatography and flow injection analysis with electrospray ionization mass spectrometry

Ovine milk proteins were analyzed both by coupling HPLC and electrospray ionization mass spectrometry (ESI-MS) and by flow injection analysis and ESI-MS detection after separation and collection of fractions from gel permeation chromatography. These methods resolved the four ovine caseins and whey proteins and made it possible to study the complexity of these proteins associated with genetic polymorphism, post-translational changes (phosphorylation and glycosylation) and the presence of multiple forms of proteins. The experimental molecular masses of ewe milk proteins were: 19 373 for κ-casein 3P; 25 616 for _s2-casein 10P; 23 411 for _s1-casein C-8P; 23 750 for β-casein 5P; 18 170 and 18 148 for β-lactoglobulins A and B; 14 152 for -lactalbumin A and 66 322 for serum albumin.     

Arsenic speciation by coupling capillary zone electrophoresis with mass spectrometry

Summary Capillary zone electrophoresis (CZE) has been coupled with mass spectrometry to enable the identification of mineral and organometallic compounds of arsenic in speciation studies. The electrophoretic effluent was introduced through a concentric interface into the mass spectrometer. Make-up liquid was added to enable electric contact at the outlet of the separation capillary and to assist the electronebulization process. After ionization, the ions were analyzed and quantified with an ion-trap detector. Optimization of the coupling conditions (geometry of the concentric interface, composition and flow rate of the sheath liquid, electronebulization and detection conditions) is described. The results show that the geometry of the concentric interface and the positioning of the outlet of the separation capillary have a critical effect on stability and sensitivity. Programming the electronebulization and detection conditions throughout the analysis enabled identification and quantification of the seven arsenic compounds of interest (neutral, and positively or negatively charged species) in less than 20 min at the ppm level. Limits of detection ranged from 0.5 to 3.3 mg L⁻¹, corresponding to amounts injected ranging from 15 to 60 pg. The linear dependence of mass spectrometric response on arsenic concentration was verified for concentrations ranging from 5 to 200 mgL⁻¹. For the two positively charged species, arsenobetaine and arsenocholine, an on-line preconcentration technique (field-amplified sample injection) enabled reduction of the detection limits by approximately one order of magnitude to 110 and 160 μgL⁻¹, respectively.     

A replaceable microreactor for on-line protein digestion in a two-dimensional capillary electrophoresis system with tandem mass spectrometry detection

We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and β-casein as model proteins.     

Capillary electrophoresis time-of-flight mass spectrometry of an opium powder

Summary Intensive work has been invested in recent years to evaluate the performance of capillary electrophoresis (CE) in forensic analysis. Tremendous progress has also been achieved in interfacing CE to sensitive and specific detection systems such as the mass spectrometer (MS). We have recently developed an electrospray time-of-flight mass spectrometer (ESI-TOFMS) for use as a detector for fast and efficient liquid phase separations. In the present paper we investigated ESI-TOFMS for the analysis of an opium powder. Both continuous infusion and CE were studied for direct sample introduction into the TOFMS and mixture separation, respectively. CEMS analysis of the opium was performed in a citrate buffer, using aqueous or mixed aqueous/organic eluents. Low fmol detection was achieved.     

Determination of chlorine species by capillary electrophoresis – mass spectrometry

The applicability of CZE with mass spectrometric detection for the determination of four chlorine species, namely chloride and three stable chlorine oxyanions, was studied. The main aspects of the proper selection of BGE and sheath liquid for the CE‐MS determinations of anions with high mobility were demonstrated, pointing out the importance of pH and the mobility of the anion in the BGE. The possibility of using uncoated fused silica capillary and common electrolytes for the separation was shown and the advantage of using extra pressure at the inlet capillary end was also presented. The linear range was found to be 1–100 µg/mL for ClO₃^? and ClO₄^?, 5–500 µg/mL for ClO₂^?, and 25–500 µg/mL for Cl^?, but the sensitivity can be greatly improved if larger sample volume is injected and electrostacking effect is utilized. The LOD for ClO₃^? in drinking water was 6 ng/mL, when very large sample volume was injected (10 000 mbar·s was applied).     

Characterization of deamidated peptide variants by micro-preparative capillary electrophoresis and mass spectrometry

Capillary electrophoresis (CE) was compared with reversed-phase liquid chromatography for its ability to separate native and deamidated peptides. CE is shown to provide superior resolution of these peptides due to its charge-based separation mechanism. Fraction collection performed using a standard CE instrument equipped with a 96-well plate permits subsequent characterization by nanospray mass spectrometric (MS) analysis. Additional in-depth analysis by MS/MS is able to provide the location of the deamidation site based on y-ion mass shifts of 1 Da.     

Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry

In this review, instrumental aspects of comprehensive two-dimensional liquid chromatography coupled with mass spectrometry are presented. The milestones of LC×LC are briefly summarized. Instrument configuration, selection of experimental conditions, the different interfaces used in the system and the current applications of LC×LC–MS systems are described.     

Identification of myosin heavy chain isoforms in porcine longissimus dorsi muscle by electrophoresis and mass spectrometry

Myosin heavy chain (MHC) isoforms have been considered as makers for muscle fiber types in relation to meat quality, whereas MHC isoforms in porcine skeletal muscle have not been fully identified. The improved technique of SDS‐PAGE and 2DE were used to separate porcine MHC isoforms. Western blotting with monoclonal antibodies including BA‐F8 (anti‐MHC slow/I), SC‐71 (anti‐MHC 2a and 2x), 10F5 (anti‐MHC 2b), and BF‐35 (anti‐MHC slow/I and 2a) and MS were used to confirm MHC migration rate and identify MHC isoforms from separated bands and spots. Up to 45% w/v of glycerol, 8% w/v of acrylamide content, and 25 h of electrophoretic time at 70 V allowed a clear separation of MHC isoforms. Major MHC isoforms such as slow, 2a, 2x, and 2b were clearly separated by SDS‐PAGE. A total of 23 MHC spots were separated and identified by 2DE and MS. Therefore, four MHC isoforms such as slow/I, 2a, 2x, and 2b could be identified by the improved SDS‐PAGEtechnique, 2DE and MS. Therefore, these techniques allow more accurate and accessible analysis in muscle fiber typing and in relationship between MHC isoforms, muscle fiber characteristics, and pork quality.