阿尔兹海默症血清多肽组生物标志物研究

采用个体样品单独分析的方式分析, 比较9个健康对照者、10个阿尔兹海默症(Alzheimer′s disease, AD)患者和12个认知功能障碍(Mild cognitive impairment, MCI)患者的血清多肽组分析结果, 以寻找潜在的AD病生物标志物.结果表明, 高强度的α-2-巨球蛋白肽段VGFYESDVMGR与AD病晚期阶段密切相关, 而载脂蛋白 C-Ⅲ、组蛋白H1.2和组蛋白H1.4的大量降解, 则与中早期AD和认知功能障碍相关联;载脂蛋白 C-Ⅲ和组蛋白H1的降解肽段具有明显的阶梯序列特征, 但在不同样本中的分布具有一定偶然性.AD病发展的晚期与中早期的血清多肽组特征不同, 这4种蛋白质的降解有可能成为AD病潜在的生物标志物.研究结果也证明了, 归属于纤维蛋白原α链、胸腺素β-4和斑联蛋白等蛋白质的肽段是所有血清样本中的优势肽段.本研究提出了利用血清多肽组学方法辅助诊断AD病的方法, 为临床大规模验证提供了依据和参考.     

基于超高效液相色谱-高分辨质谱的多肽组学技术用于人参不同部位多肽的差异分析

采用基于超高效液相色谱-高分辨质谱的多肽组学技术对人参主根、支根、须根和芦头的多肽谱进行全面分析,旨在评价人参不同形态区域多肽表达的异同。本研究共表征62个数据库中已收录的人参多肽。结果表明,人参不同部位均富含多肽类成分。多肽组学研究发现,人参主根和支根、芦头与须根之间多肽含量具有显著差异,从鉴定到的多肽中共发现25个稳定表达的已知潜在多肽标志物。其中多肽种类及含量在主根与其他部位间差异最显著,为主根与非主根药效差异研究提供了新思路。本研究揭示了人参多肽结构多样性及人参不同部位人参多肽表达的异同,对人参化学特征评价具有重要意义,为人参质量控制和合理应用提供了化学依据。     

五味子治疗大鼠糖尿病肾病作用机制的血清代谢组学研究

采用基于超高效液相色谱与串联四级杆飞行时间质谱仪(Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry,UPLC/Q-TOF-MS)联用技术的代谢组学方法,通过分析大鼠血清内源性代谢物的变化,研究五味子治疗糖尿病肾病的作用机制。利用高脂高糖饲料喂养并腹腔注射链脲佐菌素(STZ)建立糖尿病大鼠模型。给药12周后,采用试剂盒方法测定尿蛋白、尿肌酐的含量,结果表明五味子水提取物可以显著降低模型动物的尿蛋白含量(p<0.05),对糖尿病大鼠肾病并发症具有一定的改善作用。采用UPLC/Q-TOF-MS方法分析了五味子对糖尿病肾病大鼠的血清代谢轮廓,分析了健康组、模型组和五味子给药组的大鼠血清,采用偏最小二乘法-判别分析(Partial least squares discriminant analysis,PLS-DA)进行数据分析。PLS-DA得分图显示健康组、模型组和五味子组的代谢轮廓有显著差别,根据正交偏最小二乘法-判别分析(Orthogonal partial least squares discriminant analysis,OPLS-DA)载荷图筛选,将对各组分离贡献大的化合物的串联质谱分析数据,经Human Metabolome Database(HMDB)等数据库检索,进行质谱信息匹配,鉴定出黄尿酸、油酰胺、棕榈酰胺、尿酸、5-羟基己酸、硫酸对甲酚、对甲酚葡萄糖苷酸7种内源性代谢物为生物标记物。研究结果表明五味子通过影响色氨酸代谢、嘌呤代谢、肠内菌代谢、脂肪酸代谢等通路对糖尿病肾病发挥治疗作用,其中嘌呤代谢、肠内菌代谢通路可能是五味子发挥治疗作用的重要途径。     

格列美脲治疗的2型糖尿病大鼠的尿液代谢组学研究

采用快速高分离度液相色谱-质谱技术(RRLC-MS)检测经格列美脲治疗的2型糖尿病大鼠尿液中代谢物的变化, 对糖尿病组、 给药组和健康对照组大鼠的尿液代谢物谱进行了分析. 采用主成分分析(PCA)对3组大鼠进行分类并寻找潜在生物标记物. 结果表明, 3组大鼠的尿液代谢物谱得到了很好的区分, 发现并鉴定了2个潜在生物标记物, 分别为4-脱氧三羟基丁酸和4-胍基丁酸. 格列美脲对2型糖尿病大鼠的药物作用可能体现为对氨基酸代谢的调节作用.     

基于UPLC-oaTOF-MS的糖尿病及糖尿病肾病的代谢组学研究

以糖尿病患者、糖尿病肾病患者和正常人的血清为研究对象, 采用超高效液相色谱-飞行时间质谱建立其代谢指纹图谱, 并结合主成分分析进行模式识别, 实现患者和正常人的区分, 并试图发现潜在的标志物.     

血清多肽组及其个体差异的纳升液相色谱-高分辨串联质谱分析

分析和比较疾病组及健康对照组的混合样品是血清多肽组生物标记物研究的常用方法,但对健康个体多肽组的差异和共性关注较少.本研究利用纳升液相色谱-高分辨四级杆飞行时间质谱鉴定健康人混合血清样品(20例)的多肽组,阐明血清多肽组的分子量分布等一般特征,进而选取6例个体样品单独分析并与混合样品的分析结果进行比较,说明正常健康样品之间的个体差异和共同成分.结果表明,可鉴定序列的血清多肽组的分子量范围在7000 Da以下,纤维蛋白原α链等蛋白质所属肽段的检出频率最高,肽段在蛋白质水平上分布具有不均一性,排在前10％的蛋白质占据了约50％的总肽段,而后40％的蛋白质只有1条检出肽段.此外,在所有样品中都检测到了来自于8个蛋白质的12个共同肽段,检测到了N端乙酰化、氨基酸氧化、磷酸化、脱氨化和脱水等翻译后修饰和明显的阶梯序列现象.本研究在肽段序列水平分析了血清多肽组的基本特征和个体差异,可为血清多肽组生物标志物研究提供参考.     

代谢组学分析技术及其在几类重大疾病研究中的应用

代谢组学正成为生物医学研究领域的新热点。各种代谢组学分析技术各有优缺点,配有低温探头的核磁共振、混合型串联傅里叶变换质谱以及多种联用技术将成为代谢组学研究的关键技术。目前,大量多维代谢组学数据的分析方法和专用软件急待开发完善。代谢组学在肿瘤,老年痴呆症、心血管疾病、肝肾脏类疾病等研究中的应用取得一定进展,疾病代谢组学具有良好的发展前景。     

尿液多肽组的时间可变性和相对稳定性研究

尿液多肽组可反映机体的病理变化,提供潜在的生物标志物信息.目前,尿液多肽组研究通常采用晨尿,其成份是否随采样时间变化尚不清楚.本研究采集了3个健康志愿者的不同时间点的尿液,经氧化石墨烯-磷酸镧纳米复合材料(LaGM)方法分离后,利用LC-MS/MS进行序列鉴定.发现在不同取样时间下,自同一个人的尿液多肽组成分存在差异,...     

基于UPLC-Q/TOF MS技术的痛风患者血清代谢组学研究

采用超高效液相色谱-飞行时间质谱联用技术（UPLC-Q/TOF MS）对恩施土家族苗族自治州60例健康志愿者（对照组）和65例痛风患者（痛风组）的血清样本建立代谢图谱,基于主成分分析及正交偏最小二乘判别分析对所得数据进行模式识别,并结合变量权重投影分析及火山图筛选出痛风患者的血清代谢标志物。通过数据分析和数据库检索,共筛选出63种差异代谢物,其中27种代谢物显著上调（P <0.05）,36种代谢物显著下调（P <0.05）,主要包括甘油磷脂类、氨基酸类及胆碱等成分。首先,对以上差异代谢物进行受试者工作特征曲线（ROC）分析,其中曲线下面积（AUC）大于0.8的14种代谢物是诊断效能较好的代谢物;然后对筛选的63种差异代谢物进行代谢通路富集分析,以Impact> 0.1且P <0.05为标准,得到影响最大的代谢通路主要有甘油磷脂代谢、醚性脂质代谢、亚油酸代谢、半胱氨酸和蛋氨酸代谢、花生四烯酸代谢及戊糖和葡萄醛酸的互相转化等。综上,痛风患者和健康对照人群的血清代谢水平有明显差异,差异代谢物的鉴定为痛风的发病机制和早期筛查提供了实验依据。     

人血清内源性多肽无标记定量分析新策略

多肽组学(Pepti domics)以其在疾病标志物发现中所起的重要作用正受到越来越多的关注.然而,目前尚缺少针对多肽组学的高效定量方法.本文建立了一种基于纳升液相色谱与四极杆飞行时间质谱联用(μLC-Q-TOF-MS/MS)系统的色谱峰面积无标记多肽定量方法.该方法首先对两组样品中的多肽进行相对定量,然后选取相对量大于2或小于0.5的多肽,用纳升液相色谱与串联二级质谱(μLC-MS/MS)的选择性数据依赖模式(DDA)进行序列鉴定.将不同量标准样品牛血清白蛋白(BSA)酶解液加入到相同量的正常人血清样品中,然后用该方法进行定量分析,得到的定量结果平均相对偏差为6.42%.该方法分别被用于健康人血清与肝细胞癌(HCC)以及乳腺癌病人血清中内源性多肽的定量比较分析,并成功鉴定了血清中一些与HCC和乳腺癌相关的内源性多肽.由于Q-TOF-MS/MS的高分辨率和宽质量检测范围,价态高达+5和分子量超过4000的多肽也可被可靠地进行定性和定量分析.实验结果进一步表明在多肽组学分析中μLC-Q-TOF-MS/MS比传统的MALDI-TOF-MS具有更高的检测灵敏度.     

A strategy with label-free quantification of the targeted peptides for quantitative peptidome analysis of human serum

Peptidomics draws more and more attention in discovering useful biomarkers for early diagnosis of disease. However, there is lack of efficient quantification strategy in peptidome analysis. In this study, a strategy with label-free quantification of the targeted endogenous peptides based on peak intensity using μUPLC-Q-TOF-MS/MS was developed for quantitative peptidome analysis of human serum. Different amounts of standard BSA tryptic digesting peptides were added into the same serum extracts for evaluation...     

Quantitative analysis of phytoestrogens in kudzu-root, soy and spiked serum samples by high-pressure liquid chromatography

A sensitive and reliable HPLC method that allows simultaneous quantification of phytoestrogens extracted from kudzu-root and soy preparations, and serum samples has been developed. Kudzu-root and soy preparations were mixed with 5 microg flavone and 15 microg rutin (internal standards) and the phytoestrogens were extracted by using solid-phase (C18) extraction cartridges. Blank or spiked serum samples were extracted by using either C18 cartridges or trichloroacetic acid-methanol extraction. The extracts were analyzed by the HPLC equipped with a reverse-phase (250 x 4 mm, C18) column and UV, diode-array or MS detector. A linear gradient of acetic acid and acetonitrile provided excellent separation of glycoside and aglycone-phytoestrogens from kudzu root and soy preparations. The C18 cartridge extraction of serum yielded excellent recovery of both glycoside- and aglycone-phytoestrogens, while the trichloroacetic acid-methanol extraction yielded excellent recovery of glycoside but poor recovery of aglycone compounds. UV and MS detectors were suitable for phytoestrogen analysis in plant and serum samples, while the diode-array detector was suitable for generating the UV absorbance curve for phytoestrogens.     

Quantitative analysis of urinary endogenous markers for the treatment effect of Radix Scutellariae on type 2 diabetes rats

The effect of Radix Scutellariae treated on type 2 diabetic rats has been investigated by a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) based urinary quantitative approach. In this research, multiple reactions monitoring mode of MS/MS in LC-MS/MS analysis was used to quantitatively analyze the concentrations of 7 endogenous compounds in urine of normal control group, type 2 diabetic model group and Radix Scutellariae-treated group, and multivariate statistical analysis was utilized for MS data processing. The above-mentioned three groups can be distinguished via pattern recognition. The obtained results indicated that Radix Scutellariae affect the urinary metabolic profiling of type 2 diabetic rats on the polyol pathway, protein glycation reaction and amino acids metabolism pathway. According to these results, Radix Scutellariae should have the pharmacological effect on preventing or delaying the onset and progression of diabetes and its complications.     

Plasma metabolomics analysis of endogenous and exogenous metabolites in the rat after administration of Lonicerae Japonicae Flos

Lonicerae Japonicae Flos (LJF) is a typical herbal medicine and is used as a functional food. LJF, which has complex chemical compounds, has various biological effects. The global metabolomics, focusing on both the endogenous and exogenous metabolites, have not yet been investigated for LJF in normal healthy rats using LC–MS. In this study, plasma metabolomics was analyzed after the administration of LJF at different time intervals, and the exogenous metabolites were identified. Partial least squares discriminant analysis showed significant differences in chemical content in the dosed rats. Cholic acid, indoleacrylic acid, indolelactic acid, hippuric acid, N-acetyl-phenylalanine, and N-acetyl-serotonin significantly accumulated in the dosed rats. Lysophosphatidylethanolamine and lysophosphatidylcholine content, including plasmalogen, increased. There were 25 components of LJF, including 15 prototypes and 10 metabolites, that were identified. The 15 prototypes included phenolic acids, flavonoids, and iridoids, and their contents decreased with an increase in the administration time. Glucuronidation and sulfation of polyphenols were found for LJF. The exogenous glucuronide and sulfate metabolites—including dihydrocoumaric acid-sulfate, dihydrocaffeic acid-sulfate, dihydroferulic acid-sulfate, apigenin-glucuronide, apigenin-glucuronide-sulfate, isorhamnetin-glucuronide-sulfate, and others—were identified with a neutral loss of 176 and 80, respectively. The metabolic differences found in the study may serve as biomarkers of LJF consumption and promote the understanding of the mechanism of action of LJF.     

Chemiluminescent imaging analysis of interferon alpha in serum samples

Enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-catalyzed fluorescent reaction, and oxalate chemiluminescence imaging analysis have been combined to develop a sensitive, simple, and rapid method for analysis of interferon alpha (α-IFN) in human serum samples. A typical “sandwich type” immunoassay was used. Reaction of o-phenylenediamine (OPD) with hydrogen peroxide (H₂O₂), catalyzed by HRP, produced 2,3-diaminophenazine (PDA), which was detected by chemiluminescence imaging analysis with the bis(2,4,6-trichlorophenyl)oxalate (TCPO)–H₂O₂–glyoxaline–PDA chemiluminescent system. The TCPO chemiluminescent imaging system is more sensitive and the chemiluminescence quantum yield is at least five times higher than for the luminol–H₂O₂–HRP–PIP (p-iodophenol) chemiluminescent imaging system. The results showed there was a very good linear correlation between response and amount of α-IFN in the range 1.3–156.0 pg mL⁻¹ (R = 0.9991) and the detection limit was 0.8 pg mL⁻¹ (S/N=3). The relative standard deviation (n = 9) was 4.7%. The proposed method has been used for successful analysis of the amount of α-IFN in human serum. The results obtained compared well with those obtained by conventional colorimetric ELISA and luminol chemiluminescent ELISA. Figure Procedures of the proposed method     

A Zinc(II)/poly(gamma-glutamic acid) complex as an oral therapeutic for the treatment of type-2 diabetic KKAy mice

In developing new insulin-mimetic zinc(II) complexes with various ligands including a biodegradable polymer, we prepared and characterized a Zn(gamma-pga) complex in solution as well as in solid, and investigated its in vitro insulin-mimetic activity and in vivo antidiabetic effect in type-2 diabetic KKA(y) mice. The in vitro insulin-mimetic activity of the Zn(gamma-pga) complex was considerable better than that of ZnSO(4). The Zn(gamma-pga) complex normalized the hyperglycemia in KKA(y) mice within 21 d when administrated orally at doses of 10-20 mg (0.15-0.31 mmol) Zn per kg body mass for 30 d. In addition, the impaired glucose tolerance, elevated HbA(1c) levels and metabolic syndromes were significantly improved in Zn(gamma-pga)-treated KKA(y) mice relative to those administrated with saline and ZnSO(4).     

Bio-logic analysis of injury biomarker patterns in human serum samples

Digital biosensor systems analyzing biomarkers characteristic of liver injury (LI), soft tissue injury (STI) and abdominal trauma (ABT) were developed and optimized for their performance in serum solutions spiked with injury biomarkers in order to mimic real medical samples. The systems produced ‘Alert’-type optical output signals in the form of “YES-NO” separated by a threshold value. The new approach aims at the reliable detection of injury biomarkers for making autonomous decisions towards timely therapeutic interventions, particularly in conditions when a hospital treatment is not possible. The enzyme-catalyzed reactions performing Boolean AND/NAND logic operations in the presence of different combinations of the injury biomarkers allowed high-fidelity biosensing. Robustness of the systems was confirmed by their operation in serum solutions, representing the first example of chemically performed logic analysis of biological fluids and a step closer towards practical biomedical applications of enzyme-logic bioassays.     

Quantitative LIBS analysis of vanadium in samples of hexagonal mesoporous silica catalysts

The method for the analysis of vanadium in hexagonal mesoporous silica (V-HMS) catalysts using Laser Induced Breakdown Spectrometry (LIBS) was suggested. Commercially available LIBS spectrometer was calibrated with the aid of authentic V-HMS samples previously analyzed by ICP OES after microwave digestion. Deposition of the sample on the surface of adhesive tape was adopted as a sample preparation method. Strong matrix effect connected with the catalyst preparation technique (1st vanadium added in the process of HMS synthesis, 2nd already synthesised silica matrix was impregnated by vanadium) was observed. The concentration range of V in the set of nine calibration standards was 1.3-4.5% (w/w). Limit of detection was 0.13% (w/w) and it was calculated as a triple standard deviation from five replicated determinations of vanadium in the real sample with a very low vanadium concentration. Comparable results of LIBS and ED XRF were obtained if the same set of standards was used for calibration of both methods and vanadium was measured in the same type of real samples. LIBS calibration constructed using V-HMS-impregnated samples failed for measuring of V-HMS-synthesized samples. LIBS measurements seem to be strongly influenced with different chemical forms of vanadium in impregnated and synthesised samples. The combination of LIBS and ED XRF is able to provide new information about measured samples (in our case for example about procedure of catalyst preparation).     

Quantitative headspace analysis of solid samples; a classification of various sample types

Summary Volatile compounds in various solid samples have been analyzed quantitatively by equilibrium headspace gas chromatography with the technique of multiple headspace extraction (MHE). Solid samples can represent either a partition system, such as certain polymers, or an adsorption system, particularly when present as porous material. It has been found that the humidity of the sample has a strong influence on the equilibration in an adsorption system with a hydrophilic matrix and that the addition of water increases the volatility and speeds up the equilibration time. The influence of various important parameters, such as temperature, time, size and structure of the solid samples on the equilibration is demonstrated on the examples of the quantitative determination of vinyl chloride monomer in PVC, styrene in polystyrene, ethylene in polyethylene, ethylene oxide in a contact lens, halogenated hydrocarbons in coffee and residual solvents in drugs. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984