基于超高效液相色谱-高分辨质谱法的油料作物脂质组学分析

针对油料脂质分子众多、结构难识别的难题,采用超高效液相色谱-高分辨质谱对油菜籽、大豆、花生、向日葵籽、玉米5种油料作物中脂质进行分析。使用反相色谱柱ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm,1.7μm)分离脂质,超高效液相色谱-高分辨质谱仪(UPLC-Orbitrap Fusion Mass)正、负离子模式下采集Data Dependent MS~2数据,利用二级质谱碎片特征、精确分子质量、保留时间以及数据库匹配对脂质分子进行定性分析,获得脂质分子的唯一结构式。共鉴定出油料作物中132种脂质分子,包括8种甘油二酯(DG)、5种溶血磷脂酰胆碱(LPC)、2种溶血磷脂酰乙醇胺(LPE)、24种磷脂酰胆碱(PC)、18种磷脂酰乙醇胺(PE)、8种磷脂酰肌醇(PI)、1种磷脂酰甘油(PG)、1种磷脂酰丝氨酸(PS)以及65种甘油三酯(TG),并使用峰面积比进行相对含量的比较。该方法可以很好地实现脂质成分的分离,将油料作物中脂质成分的分析从脂肪酸分析水平提升到脂质分子层面,为更好地实现油料作物中脂质成分的应用提供了研究基础,并促进了脂质组学的发展。     

基于超高效液相色谱-四极杆-静电场轨道离子阱串联质谱的冷链贮藏过程中滩羊肉的脂质组学研究

该文建立了超高效液相色谱-四极杆-静电场轨道离子阱串联质谱(UHPLC-Q-Orbitrap MS/MS)结合脂质组学分析滩羊肉在冷链贮藏过程中脂质变化规律和脂质分子碎裂机理的方法。样品经异丙醇提取后,采用质谱全扫描模式和二级扫描模式对目标物质进行定性。共鉴定出48个变化显著性脂质,包括8个脂肪酰基肉碱、23个磷脂酰胆碱(PC)、3个溶血性磷脂酰胆碱(LPC)、13个磷脂酰乙醇胺(PE)、1个溶血性磷脂酰乙醇胺(LPE)。含量差异表现为部分PC、PE和脂肪酰基肉碱在前12 d短暂性升高,12 d后开始降低,而LPE和LPC在整个冷链贮藏期间表现为上升趋势。PC、PE和脂肪酰基肉碱短暂性的积累易导致大量的脂质氧化反应,进一步阐明最佳冷链时间为12 d。该方法适用于复杂基质中脂质分子的分离与定量,为肉及肉类产品在冷链贮藏过程中的脂质变化规律研究及质量控制提供了依据。     

基于鸟枪法脂质组学研究环氧酶-2抑制剂对关节炎模型血清脂代谢的干预作用

心血管疾病(CVD)是类风湿关节炎(RA)患者死亡的重要原因之一,脂代谢紊乱与CVD发生有密切关联,因而有必要对RA和药物治疗导致的脂代谢改变进行探讨。该研究采用胶原诱导法(CIA)构建关节炎模型,引入多维质谱鸟枪法开展血清脂质分析,检测了血清中105种脂质分子,发现模型大鼠体内7种磷脂酰肌醇、15种鞘磷脂、5种神经酰胺、10种磷脂酰胆碱和2种溶血磷脂酰胆碱异常上调,环氧酶-2(COX-2)抑制剂可部分修复紊乱的脂代谢,但对5种磷脂酰胆碱和1种溶血磷脂酰胆碱具有异常调控作用。该研究从脂质分子水平探讨了RA及COX-2抑制剂对脂代谢的干预作用,可以为RA的心血管风险研究提供信息。     

改性苯乙烯-马来酸酐共聚物色谱固定相用于磷脂分离分析北大核心CSCD

磷脂是重要的信号分子,磷脂的代谢与多种疾病密切相关。因此,开展磷脂的分离分析研究至关重要。苯乙烯-马来酸酐共聚物(SMA)作为一种新型两亲性交替共聚物可以插入生物膜的磷脂双分子层中,形成以膜蛋白质为中心的脂质纳米盘,对膜蛋白质和磷脂具有良好的增溶作用。本文基于“点击”反应和自由基聚合反应,将对磷脂具有良好增溶性能的SMA接枝到硅胶表面,然后以蛋氨酸甲酯盐酸盐(MME·HCl)为开环试剂,通过亲核开环反应对SMA进行修饰,制备了新型的改性SMA修饰色谱固定相(Sil-SMA-MME)。结合高效液相色谱-紫外检测法,利用酰胺类和核苷/核酸碱基类以及苯酚类3类小分子物质对填充Sil-SMA-MME色谱柱的保留机制和分离性能进行了系统评价,Sil-SMA-MME色谱柱具有典型的亲水作用保留机制,其柱效最高可达90900 N/m,并显示了良好的分离选择性。进一步结合高效液相色谱-蒸发光散射检测法,考察了Sil-SMA-MME色谱柱对磷脂样品的分离性能。二棕榈酰磷脂酰丝氨酸钠(DPPS)、二油酰磷脂酰胆碱(DOPC)、二棕榈酰磷脂酰乙醇胺(DPPE)和4种磷脂酰胆碱(PC)类标准品溶血卵磷脂(LysoPC)、二肉豆蔻酰磷脂酰胆碱(DMPC)、二硬脂酰磷脂酰胆碱(DSPC)、二棕榈酰磷脂酰胆碱(DPPC)均可实现基线分离,并且成功地实现了南极磷虾油和人血清磷脂提取物的分离分析。以上结果表明,所制备的Sil-SMA-MME色谱柱在磷脂类物质分离分析中具有良好的应用潜力。     

直接进样电喷雾串联质谱法测定草鱼肌肉组织中磷脂

建立了直接进样电喷雾串联质谱测定草鱼肌肉组织中磷脂的方法.以Bligh Dyer法提取总脂质,采用流动注射泵直接进样的方式将样品导人电喷雾离子源,利用串联三重四级杆质谱的母离子扫描和中性丢失扫描功能,通过扫描磷脂的特征性子离子或中性质量丢失实现对磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇、磷脂酰丝氨酸、磷脂酰甘油和磷脂酸六类磷脂的源内分离和鉴定.结果显示,在-定的浓度范围内,磷脂的浓度与磷脂直接进样电喷雾电离后形成准分子离子的响应值呈现良好的线性关系,回收率(67.1％～96.6％)和精密度可以满足生物样品分析的要求.采用本方法测定了草鱼肌肉组织中磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇和磷脂酰丝氨酸4类磷脂的分子种及含量.本方法前处理简单,定性和定量分析快速准确,可以应用于其它生物样本脂质组学中磷脂的分析.     

沉香的高效液相色谱-四极杆-飞行时间质谱数字化指纹图谱研究

采用高效液相色谱-四极杆-飞行时间质谱联用(HPLC-Q-TOF-MS)技术,研究构建了一种沉香数字化色谱-质谱指纹图谱的新方法。沉香药材经乙醇提取后,采用HPLC-Q-TOF-MS测定,并同时采集HPLC-Q-TOF-MS及液相色谱-紫外数据,得到液相色谱-紫外检测(HPLC-UV)色谱图和高分辨飞行时间质谱(TOF-MS)总离子流色谱图。对色谱图中的各个色谱峰进行精确质量数识别,建立数字化指纹图谱,以精确质量数结合保留时间表征沉香中的化学成分,即为每个色谱峰给出具有唯一性的数字信息,以数字化的形式反映其化学成分,并根据精确质量及同位素推算出分子式,结合二级质谱及文献资料共鉴定出30个化学成分。该方法对沉香的每种化学成分给出了类似于身份认定的数字化信息,具有唯一性,能全面反映沉香的物质成分,可为沉香的药理、药效及质量标准研究提供科学的数据。     

液相色谱-质谱用于卵巢肿瘤中磷脂轮廓的分析

卵巢肿瘤日益影响女性的健康和生活质量,其中的卵巢癌是女性三大恶性肿瘤之一,死亡率高居三者之首。因此卵巢肿瘤尤其卵巢癌是目前的一个研究热点。本研究利用液相色谱-质谱(LC-MS)联用技术对卵巢肿瘤进行磷脂轮廓分析,研究良性卵巢肿瘤(B)和卵巢癌(M)的患者血清中磷脂代谢的差异情况。首先用LC-MS采集血清中磷脂的指纹图谱,通过峰识别、峰匹配等得到峰表,然后利用正交校正的偏最小二乘法(OSC-PLS)进行多种分型,根据模型的变量重要因子(VIP)、VIP值的置信区间、S图和显著性差异检验结果等筛选有差异的磷脂。结果显示: M组和B组与正常对照(N)组比较都存在明显的磷脂代谢差异,发生改变的磷脂主要为缩醛磷脂酰乙醇胺、磷脂酰胆碱、缩醛磷脂酰胆碱、鞘磷脂和溶血磷脂酰胆碱。     

κ-卡拉胶寡糖脂的合成及其结构鉴定

以κ-卡拉胶为原料, 通过稀酸降解并结合柱层析分离得到5个寡糖单体, 经还原胺化法将其与二棕榈酸酯磷脂酰乙醇胺(DPPE)偶联首次获得了5个拟糖脂. 应用高灵敏电喷雾离子化碰撞诱导解离串联质谱(ESI-CID-MS/MS)技术确定了其序列, 证明其分别为κ-卡拉胶三糖脂、五糖脂、七糖脂、九糖脂和十一糖脂. 该研究结果为硫酸寡糖脂的合成提供了参考方法, 尤其为寡糖生物芯片的制备以及深入开展寡糖与蛋白相互作用研究提供了基础.     

台风过后网箱养殖大黄鱼恢复过程中血清代谢物变化的研究

为了考察台风影响后鱼类恢复过程中血清代谢物的变化,利用超高效液相色谱-四极杆-飞行时间质谱,电喷雾电离源分别在正负离子模式下,对超强台风"罗莎"影响后的象山港网箱养殖大黄鱼血清进行为期半个月的测定,并通过SIMCA-P软件进行主成分分析和正交偏最小二乘法辨别分析.结果表明,大黄鱼恢复过程中潜在生物标志物主要为磷脂酰胆碱和溶血磷脂酰胆碱.另外还有皮五醇和牛黄胆酸.其中,溶血磷脂酰胆碱、含高不饱和度脂肪酰基的磷脂酰胆碱(总不饱和度之和大于9)和牛黄胆酸在恢复过程中均呈现增加的趋势; 而含低不饱和度脂肪酰基的磷脂酰胆碱(总不饱和度之和小于8)和皮五醇则呈现减少的趋势.这些代谢指标物的变化,体现了大黄鱼在台风后恢复过程中,通过皮质类激素调节脂类物质代谢的结果.     

高效液相色谱-质谱法对冠心病患者血清的脂质组学研究及其临床意义

采用高效液相色谱-质谱法(HPLC-MS)对冠状动脉慢性完全堵塞(CTO)患者在不同患病阶段的血清试样进行了脂质组学研究。试验采集了CTO疑似人群、CTO患者及CTO患者经皮冠状动脉介入治疗(PCI)手术后24h和72h的血清,分别采用氯仿和体积比为25∶75的乙酸乙酯-异辛烷混合溶液提取脂质,离心分离后,上清液用于Shotgun法(MS)和多反应监测(MRM)法(HPLC-MS)分析。样本直接进样电喷雾(ESI)质谱分析,用于Shotgun法进行广谱的脂质组学分析鉴定。通过Analyst 1.6软件提取质谱数据,并通过生物信息学工具"Lipid MS Predict"确认脂质分子,总共得到1 504种脂质分子,并利用MRM法确认其中有临床意义的631个脂质分子。通过生物信息学分析,得到对于CTO具有诊断价值的包括胆固醇酯CE 16∶1和溶血磷脂酰乙醇胺LPE 18∶1在内的9个标志性脂质分子。结合临床回访结果和脂质组学研究数据证实,CTO患者手术治疗后的预后情况和磷脂酸、磷脂酰胆碱、胆固醇酯等3类脂质的含量变化有关。确定的脂质标志物应用于临床诊断早期冠心病和评估手术治疗后的风险,具有显著而明确的临床医学价值。     

Lipid analysis by thin-layer chromatography--a review of the current state

High-performance thin-layer chromatography (HPTLC) is a widely used, fast and relatively inexpensive method of separating complex mixtures. It is particularly useful for smaller, apolar compounds and offers some advantages over HPLC. This review gives an overview about the special features as well as the problems that have to be considered upon the HPTLC analysis of lipids. The term "lipids" is used here in a broad sense and comprises fatty acids and their derivatives as well as substances related biosynthetically or functionally to these compounds. After a short introduction regarding the stationary phases and the methods how lipids can be visualized on an HPTLC plate, the individual lipid classes will be discussed and the most suitable solvent systems for their separation indicated. The focus will be on lipids that are most abundant in biological systems, i.e. cholesterol and its derivates, glycerides, sphingo- and glycolipids as well as phospholipids. Finally, a nowadays very important topic, the combination between HPTLC and mass spectrometric (MS) detection methods will be discussed. It will be shown that this is a very powerful method to investigate the identities of the HPTLC spots in more detail than by the use of common staining methods. Future aspects of HPTLC in the lipid field will be also discussed.     

Analysis of cancer cell lipids using matrix‐assisted laser desorption/ionization 15‐T Fourier transform ion cyclotron resonance mass spectrometry

A combination of methodologies using the extremely high mass accuracy and resolution of 15‐T Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometry (MS) was introduced for the identification of intact cancer cell phospholipids. Lipids from a malignant glioma cell line were initially analyzed at a resolution of >200 000 and identified by setting the mass tolerance to ±1 mDa using matrix‐assisted laser desorption/ionization (MALDI) 15‐T FT‐ICR MS in positive ion mode. In most cases, a database search of potential lipid candidates using the exact masses of the lipids yielded only one possible chemical composition. Extremely high mass accuracy (<0.1 ppm) was then attained by using previously identified lipids as internal standards. This, combined with an extremely high resolution (>800 000), yielded well‐resolved isotopic fine structures allowing for the identification of lipids by MALDI 15‐T FT‐ICR MS without using tandem mass spectrometric (MS/MS) analysis. Using this method, a total of 38 unique lipids were successfully identified. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterisation of membrane phospholipids and glycolipids from a halophilic archaebacterium by high-performance liquid chromatography/electrospray mass spectrometry

Combined high-performance liquid chromatography and electrospray mass spectrometry (LC/ES-MS) has been used for direct characterisation of the polar membrane lipids in total lipid extracts from Halobacterium salinarium, a species of halophilic archaebacterium. The principle phospholipids found were the diphytanyl archaeol phosphatidylglycerol and diphytanyl archaeol phosphatidylglycerolphosphate methyl ester. The application of LC/ES-MS revealed the additional presence of diphytanyl archaeol phosphatidylglycerol sulphate The extracts also contained an archaeol glycolipid, initially detected in preliminary offline ES-MS studies, which was further characterised by LC/ES-MS and by product ion tandem mass spectrometry (MS/MS) as a sulphate ester of diglycosyl-2,3-di-O-phytanyl-sn-glycerol. Whilst archaeol phospho- and glycolipids containing a (C(20)-C(20))-isopranyl glycerol ether core predominated, LC/ES-MS of the extracts from Halobacterium salinarium indicated the presence of an analogue containing one double bond in its isoprenyl ether core as a minor component of the phosphatidylglycerolphosphate methyl ester fraction, providing a further example of the previously recognised existence of isoprenologues of diphytanyl archaeols which occur as minor components of archaebacterial membrane lipids. The value of these techniques in compositional analysis of archaebacterial lipid extracts is discussed.     

Lipid fingerprinting of gram-positive lactobacilli by intact--matrix-assisted laser desorption/ionization mass spectrometry using a proton sponge based matrix

A method of direct lipid analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) in intact membranes, without prior extraction/separation steps, is described. Here, we demonstrate the efficacy of a strong base, 1,8-bis(dimethylamino)naphthalene (DMAN; proton sponge), as a novel matrix for MALDI-time-of-flight (TOF) MS analysis of whole cell bacteria. Initially, individual acidic low-molecular-weight analytes such as standard free fatty acids and phospholipids were analyzed using DMAN as matrix. Clear negative-mode MALDI-TOF MS spectra of all analytes show only deprotonated analyte signals at a low picomole limit of detection with the complete absence of matrix-related signals. These results indicate that DMAN represents a suitable matrix for MALDI-TOF MS analysis of mixtures of complex lipids as the intact membranes of microorganisms. DMAN was successfully applied to the analysis of Lactobacillus sanfranciscensis and L. plantarum microorganisms. Different components were sensitively detected in a single spot, including 16:0, 18:2, 18:3, and 21:0 free acids, glycolipids, phosphatidylglycerols (PGs) and cardiolipins. This method might be of general application, offering the advantage of quickly gaining information about lipid components of other gram-positive bacterial membranes.     

Extraction of methyl xanthines and their UHPLC–DAD determination in consumable beverages used in Eastern province of Saudi Arabia

Coffee and tea are the most widely consumed beverages worldwide. However, the consumer may be unaware of the exact amount of methyl xanthine (MX, i.e. caffeine [C], theobromine [TB] and theophylline [TH]) consumed, as most of the products do not list the proper amounts. This may lead to serious risks including cardiovascular, kidney and stimulant effects. The aim of the study was to determine the MX amount in ready-to-use beverages (coffee and tea) collected from various outlets in the city of Al-Khobar, Saudi Arabia. Forty different samples of espresso, black coffee and red tea were collected. A fast, reliable and efficient UHPLC–DAD method was developed and validated for MX determination. Total lipids were extracted and fractionated in order to determine glycolipids, phospholipids and neutral lipids. The r² value for the method was 0.980–0.988 in a linearity range of 0.5–200 ppm. The range for MX (C [0.02–2.39 mg/ml], TB [0.00–0.10 mg/ml] and TH [0.00–0.004 mg/ml]) and total lipids was 1–5 g. The amount of glycolipids (3.1 g) was higher among the lipid fractions followed by phospholipids (1.8 g) and neutral lipids (0.25 g). In general, espresso beverages (20–30 ml) contained high amounts of MX whereas black coffee beverages contained high amount of lipids. Most of the beverages expressed C, TB, TH, lipids or their fractions; however, the product with high amounts of MX and lipids at the same time was espresso (brands Chemistry and Wogard). Although the MX and lipid levels in these beverages well below the allowed limits, care must still be taken, especially when using the beverages with high serving volumes (200–250 ml) or coffee prepared via the filter method i.e. black coffee, using a high temperature for a longer time.     

Facile distinction of neutral and acidic tetraether lipids in archaea membrane by halogen atom adduct ions in electrospray ionization mass spectrometry

Calditocaldarchaeol (neutral tetraether lipid) from Sulfolobus acidocaldarius (acidothermophilic archaea) and intact total lipid from the thermoacidophilic archaea Sulfolobus sp. was examined by electrospray ionization time-of-flight mass spectrometry in the negative-ion mode using high resolution. When the sample was injected as a solution in a 3:1 mixture of methanol (MeOH) and chloroform (CHCl(3)) using an infusion system, the total ether lipid afforded molecular-related ions as [M - H](-) for acidic polar lipids containing a phosphoric or sulfuric group, and as [M + Cl](-) ion for neutral glycolipids. The attachment of chloride was confirmed by the observation of [M + Br](-) ion, instead of [M + Cl](-) ion, when a 3:1 mixture of MeOH and CHBr(3) was used in place of MeOH-CHCl(3) as the solvent. The composition of tetraether neutral glycolipids that are different from each other only in the number of five-membered rings in the isoprenoid chain was determined on the basis of the isotope-resolved mass spectrum of [M + Cl](-) ions. As for acidic tetraether lipids, molecular-related ions [M - H](-)) were not observed when the 3:1 MeOH-CHBr(3) mixture was used as the solvent. These results together afforded a facile method of distinguishing neutral from acidic tetraether lipids in intact total lipids of acidothermophilic archaea. This method was applied to determine the difference of the number of five-membered rings in isoprenyl chains of neutral tetraether glycolipids yielded by the Sulfolobus sp. grown at different temperatures. Discrimination of neutral tetraether glycolipids from acidic tetraether lipids in the total lipids obtained from Thermoplasma sp. was also achieved by this method.     

Analysis of brain lipids by directly coupled matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high-performance thin-layer chromatography

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a soft ionization MS technique providing only minor fragmentation of the analyte. Therefore, the method is basically suitable for mixture analysis, although the ion yields strongly depend on the basicity/acidity of the analyte in relation to the applied matrix. Accordingly, less sensitively detectable compounds may be suppressed by more sensitively detectable compounds. Thus, separation of the mixture into the individual compounds is normally indispensable. This paper demonstrates the capabilities and limitations of a direct, simple, and inexpensive MALDI-high-performance thin-layer chromatography (HPTLC) coupling for the analysis of a crude lipid extract from porcine brain. Brain lipids were chosen because they represent a rather complex mixture and are of currently significant research interest. It was found that normal-phase HPTLC-separated lipids can be easily characterized by direct MALDI-TOF-MS analysis with sufficient resolution to allow the assignment of virtually all lipid classes, even rather minor species such as phosphorylated phosphoinositides or complex glycolipids as gangliosides. Advantages and disadvantages of this approach are discussed.     

Separation of lipid classes from plant tissues by high performance liquid chromatography on chemically bonded stationary phases

The wide range of lipid classes found in plant tissues, including simple lipids, glycolipids, and phospholipids, have been resolved by gradient high performance liquid chromatography with evaporative light-scattering detection. The lipids from potato tubers were used to optimize the separations. Adsorption chromatography with silica gel gave disappointing results for the plant glycolipids. Much better separations were obtained with chemically bonded stationary phases, e.g. those with propionitrile or polymeric vinyl alcohol as the functional moiety. With careful calibration, good reproducibility was possible. Only highly acidic lipids such as phosphatidylserine still present some problems.     

Lipids ofViburnum opulus seeds

Lipid components of the seeds ofViburnum opulus (Caprifoliaceae family) were investigated. The neutral lipids consist of eight classes, the glycolipids consist of three classes, and the phospholipids contain seven classes. The fatty-acid contents of all of the acyl-containing lipids were determined. The 18∶2 fatty acid is the main component of all the lipid fractions. The content of saturated acids is greater in the glycolipids and phospholipids. The lipophilic components, higher fatty alcohols and sterols, were identified.     

Profiling and quantification of Drosophila melanogaster lipids using liquid chromatography/mass spectrometry

We present here the findings of global profiling of Drosophila lipids using liquid chromatography/tandem mass spectrometry (LC/MS/MS) on an LTQ-Orbitrap instrument. In addition, we present a multiple reaction monitoring (LC-MRM) method for the absolute quantification of the major phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids of Drosophila. Using both normal- and reversed-phase LC followed by accurate mass analysis and MS/MS on an LTQ-Orbitrap instrument, we evaluated the lipid composition of the fruit fly Drosophila melanogaster. A total of 74 lipid species were identified consisting of glycerphospholipids belonging to the PE, PC, phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS) classes including several plasmanyl PE species, as well as triacylglycerides, cardiolipins, ceramides, and PE ceramides. Individual PE and PC phospholipids were then quantified using an LC-MRM approach. Reversed-phase chromatography followed by monitoring on a QTrap 4000 instrument of 21 MRM transitions combined with calibration curves constructed using internal standards enabled the absolute quantification of 28 PE and PC lipid species with limits of quantification of 3 and 5 pg/μL, respectively. Internal standards accounted for the differences in ionization efficiencies of PE and PC phospholipids, facilitating more accurate lipid abundance measurements. The method presented here builds on previous Drosophila work by making the quantification of absolute lipid abundance possible and will be of interest to scientists who study variation and changes in the degree of unsaturation, fatty acid carbon length, and head-group concentration among individuals of different genotypes in response to environmental, genetic, or physiological perturbation in small insects. It will also be particularly useful to biologists interested in adaptation and acclimation of cellular membranes in response to thermal heterogeneity.