超高效液相色谱-串联质谱法测定血浆中烷基间苯二酚

目的：建立超高效液相色谱-串联质谱法测定血浆中2种全麦食品生物标志物烷基间苯二酚（ARs）的方法。方法：血浆加入内标,经丙酮提取,离心取上清液过滤,滤液吹干异丙醇定容,使用实心核色谱柱（2.1mm×100mm,1.6μm）为分离柱,以异丙醇∶乙腈=30∶70为流动相,等度洗脱,质谱采用电喷雾负离子模式,多反应监测模式进行检测,用内标法定量。结果：2种ARs在2~200ng/mL浓度范围内线性良好,r2>0.998 0。方法检出限为0.1ng/mL,定量限为0.3ng/mL,加标回收率为88.2%~110%,相对标准偏差为2.48%~13.2%（n=6）。结论：该方法快速准确、灵敏度高、前处理简单,能够有效测定血浆中2种ARs的含量。     

超高效液相色谱-电喷雾串联四级杆质谱鉴定灵芝中脂质成分

脂质是灵芝重要活性成分之一,但目前对灵芝胞内脂质成分的构成研究甚少。本研究采用UPLC-ESI-MS/MS技术,对灵芝发酵菌体的胞内脂质构成进行分析。结果显示,灵芝细胞中共鉴定到296种脂质,其中甘油酯112种、磷脂148种、鞘脂34种和甾醇2种;甘油酯和磷脂分别占总脂质的44.70%和38.06%,鞘脂和甾醇分别占总脂质的17.08%和0.16%。分析甘油酯中主要成分为甘油三酯,占甘油酯总含量的67.36%;磷脂中主要成分为磷脂酰乙醇胺,占磷脂总含量的62.64%;鞘脂中主要成分为神经酰胺,占鞘脂总量的60.33%;此外,本研究检测出27种游离脂肪酸,其中20种为不饱和脂肪酸,相对含量为65.59%;7种为饱和脂肪酸,相对含量为34.41%。本研究系统性地分析了灵芝细胞中的脂质构成,为进一步开展灵芝细胞中脂质相关研究奠定基础。     

超高效液相色谱-串联质谱法检测小菜蛾血清中的β-蜕皮激素

[目的]研究建立可以准确检测小菜蛾血清中痕量β-蜕皮激素的超效液相色谱-串联质谱(UPLC-MS/MS)分析法.[方法]小菜蛾血清β-蜕皮激素样品用乙腈提取并进行蛋白沉淀,以C18色谱.柱分离,经UPLC-MS/MS检测,以芸苔素内酯为内标物,内标法定量.[结果]结果表明,在0.5-50 μg/L浓度范围内β-蜕皮激素...     

超高效液相色谱-串联质谱法同时测定血清中维生素A、E的方法研究

目的：建立超高效液相色谱 串联质谱（UPLC MS/MS）同时测定血清中维生素A、E的方法。方法：取标准系列工作溶液、空白替代血清样品（4% BSA牛血清白蛋白溶液）各50 μL,经硫酸锌、沉淀剂（甲醇/乙腈=50/50,V/V）沉淀蛋白,振荡,12 000 r/min离心5 min,取上清,低压离心挥干,复溶振荡,12 000 r/min离心5 min,取上清待测。采用Waters BEH Phenyl色谱（2.1 mm×100 mm,1.7 μm）分离,以0.1%甲酸 水溶液和0.1%甲酸 甲醇溶液为流动相等度洗脱,电喷雾（ESI）正离子模式、多反应监测模式（MRM）下检测,内标法定量。结果：UPLC MS/MS检测血清维生素A、α 维生素E、β 维生素E线性关系良好,相关系数分别为0.999 3、0.999 3、0.999 7;维生素A、α 维生素E、β 维生素E定量限分别为0.213、0.240、0.070 ng/mL,检出限分别为0.064、0.072、0.021 ng/mL;维生素A、E在低、高浓度加标回收率范围分别为89.7~107.4、97.6~118.1,RSD分别为2.45%~3.43%、1.48%~5.40%。结论：本研究建立的人血清维生素A、E超高效液相色谱 串联质谱法灵敏度高、准确稳定、重现性好,对血清中维生素 A 和维生素 E 的测定具很好的的应用价值。     

分散固相萃取剂-液相色谱串联质谱法测定鸡肉和鸡蛋中氟苯尼考和氟苯尼考胺残留

建立分散固相萃取剂-液相色谱串联质谱法(HPLC-MS/MS)同时检测鸡肉及鸡蛋中氟苯尼考和氟苯尼考胺的方法.样品用乙腈提取,C18分散固相萃取填料净化,乙腈饱和的正己烷脱脂,电喷雾离子源正负模式切换,HPLC-MS/MS多反应监测(MRM),同位素内标法定量.氟苯尼考和氟苯尼考胺线性范围分别为0.1 ng/mL～2....     

鳞杯伞α-半乳糖苷酶的提取纯化及酶学性质研究

采用离子交换层析和凝胶过滤层析对鳞杯伞子实体中的α-半乳糖苷酶进行纯化,得到了一种分子量为50 kDa的α-半乳糖苷酶,命名为CSG。纯化后的CSG纯化倍数为891.46倍,比活力为54.78 U/mg,得率为0.71%。通过BLAST比对液相色谱-串联质谱(LC-MS/MS)获得其肽段,发现其为GH27家族的α-半乳糖苷酶。CSG的最适pH为3.0,最适温度为50 ℃。在酸性范围pH 2.2-7.0和温度范围4-30 ℃有较好的稳定性。Mn²⁺、Cd²⁺、Cu²⁺对CSG有较强的抑制作用。半乳糖和蜜二糖对CSG的抑制类型为混合型抑制。化学修饰剂N-溴代琥珀酰亚胺显著降低CSG的活力,碳二亚胺对CSG具有显著的激活作用。该酶具有良好的蛋白酶抗性,且对棉子糖家族寡糖(RFOs)、瓜尔豆胶和赤槐豆胶均表现出良好的水解作用。     

壳聚糖和ε-聚赖氨酸处理对双孢蘑菇贮藏过程中品质的影响

以延长双孢蘑菇货架期为目标,探讨壳聚糖和ε-聚赖氨酸处理对采后双孢蘑菇在4 ℃贮藏过程中生理特性、营养品质和贮藏特性的影响。结果表明：与对照组双孢蘑菇相比,壳聚糖与ε-聚赖氨酸6:4复配溶液处理能够有效抑制双孢蘑菇表面微生物的生长和多酚氧化酶活性的增加,保持双孢蘑菇子实体较高的L*值和硬度,延缓双孢蘑菇的质量损失和细胞膜透性的升高,减少双孢蘑菇子实体的腐烂,保持较高商品率。在4 ℃条件下,壳聚糖与ε-聚赖氨酸6:4复配溶液对双孢蘑菇的保鲜性能最优,能够有效保持双孢蘑菇的商品品质和延长其贮藏时间。     

建立高效液相色谱-串联质谱检测细胞内腺苷酸浓度的方法及其应用

细胞内腺苷酸浓度变化是细胞能量代谢改变的感应器,建立高效液相色谱-串联质谱法检测细胞内腺苷酸浓度的方法有助于监测药物对细胞能量代谢的影响．用含有Na-EDTA的高氯酸溶液超声裂解细胞．采用超高效HSS T3色谱柱 (2.1 mm ×100 mm, 1.8 μm),以8 mmol/L N, N-二甲基己胺(DMHA)水溶液和乙腈为流动相进行梯度洗脱,采用正离子模式质谱检测,在多反应监测(MRM)模式下进行定性定量分析．结果表明,AMP、ADP和ATP分别在(0.1814～14.5164) μmol/L、(0.2342～18.7354) μmol/L和(0.2003～16.0260) μmol/L线性范围内具有良好的线性关系,其相关系数分别为0.9984、0.9964和0.9990．AMP、ADP和ATP的检出限(LOD,S/N>3)分别为1.9291、1.8794 和166.5 nmol/L,定量限(LOQ,S/N>10)为1.9632、1.9672和185.6 nmol/L,且加标回收率为81.8% ～107.8%,相对标准偏差小于7.55%．AMP、ADP和ATP的日内偏差(RSD)分别为6.16%、5.13%和7.66%,日间偏差(RSD)分别为6.36%、2.74%和6.77%．该方法快速、简单、灵敏,能满足细胞内AMP、ADP和ATP含量的检测要求．通过检测分析在不同浓度高良姜挥发油作用下人肺癌A549细胞内AMP、ADP和ATP含量变化,结果显示细胞总的腺苷酸水平和能荷呈浓度依赖性下降,且当浓度达到500 mg/L时ATP/TAN明显下降,而A549细胞中AMP/ATP比例水平呈浓度依赖性增加．这提示高良姜挥发油可通过影响细胞能量代谢抑制细胞增殖．     

高效液相色谱-串联质谱分析微量格尔德霉素类似物

安莎类抗生素例如利福霉素和安丝菌素,通常由一组化学结构相似的组分组成。格尔德霉素为苯安莎类抗生素,已经发现4个组分。本研究采用高效液相色谱-串联质谱方法对格尔德霉素(GDM)制品中的微量组分进行了分析,发现5个新的和1个已知的GDM类似物。依据质谱数据、结合GDM生物合成机制,对6个GDM类似物的化学结构进行了推测:分子式为C29H42N2O10的新化合物3个,分别为GDM安莎链上C2-C3、C4-C5和C8-C9之间的C-C双键变为单键并同时单羟基化的GDM衍生物;分子式为C28H38N2O8的新化合物2个,其中1个为17(或12,或4)-去甲氧基格尔德霉素,另1个为4,5-双氢-10,11-脱水-17-去甲基-17-羟基格尔德霉素;分子式为C29H42N2O9的已知化合物1个,为4,5-双氢格尔德霉素。这些GDM类似物的发现有助于加深对GDM生物合成的认识,并对通过基因阻断、组合生物合成技术获得GDM衍生物的研究有启示作用。     

基于超高效液相色谱-串联质谱技术的柑橘黑点病菌(Diaporthe citri)发育关联代谢物分析

【目的】柑橘黑点病是柑橘间座壳菌(Diaporthe citri)引起的真菌性病害,是危害柑橘的重要病害之一,D. citri在生长发育过程中经历菌丝生长(10 d, T1)、分生孢器形成(20 d, T2)和分生孢器产孢(30 d, T3)三个阶段。通过不同发育阶段代谢组分析,挖掘病原菌发育过程中标记物、关键代谢物,为黑点病菌产孢机制、代谢调控等深入研究提供依据。【方法】利用超高效液相色谱-串联质谱(ultra performance liquid chromatography/tandem mass spectrometry, UPLC-MS/MS)技术分析了D. citri发育过程中的代谢变化,采用主成分分析(principal component analysis, PCA)和正交偏最小二乘判别分析(orthogonal partial least squares discriminant analysis, OPLS-DA),筛选出了显著差异代谢物并进行了KEGG (Kyoto encyclopedia of genes and genomes)富集分析。【结果】D. cit...     

Ultrastructural localization of integrin subunits beta4 and alpha3 within the migrating epithelial tongue of in vivo human wounds

Subsequent to wounding, keratinocytes must quickly restore barrier function. In vitro wound models have served to elucidate mechanisms of epithelial closure and key roles for integrins alpha6beta4 and alpha3beta1. To extrapolate in vitro data to in vivo human tissues, we used ultrathin cryomicrotomy to simultaneously observe tissue ultrastructure and immunogold localization in unwounded skin and acute human cutaneous wounds. Localization of the beta4 integrin subunit in unwounded skin shows dominant hemidesmosomal association and minor basal keratinocyte lateral filopodic cell-cell expression. After wounding, beta4 dominantly localized to cytokeratin-rich regions (trailing edge hemidesmosomes) and minor association with lamellipodia (leading edge). beta4 colocalizes with alpha3 within filopodia juxtaposed to wound matrix, and increased concentrations of beta4 were found in cytoplasmic vesicles within basal keratinocytes of the migrating tongue. alpha3 integrin subunit dominantly localized to filopodia within basal keratinocyte lateral cell-cell interfaces in unwounded skin and both cell-cell and cell-matrix filopodic interactions in wounded skin. This study indicates that beta4 interacts with the extracellular environment through both stable and transient interactions and may be managed through a different endosomal trafficking pathway than alpha3. alpha3 integrin, despite its ability to respond to alternate ligands after wounding, does so through a single structure, the filopodia.     

Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and “dilute and shoot” procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.     

EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and beta-lactam cleaving activity

Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and beta-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of beta-lactamases and DD-peptidases. The chemical reactivities of esters and beta-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying beta-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C beta-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "beta-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to beta-lactamases, EstB shows no beta-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous beta-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus beta-lactam cleavage.     

The brown seaweed Sargassum hemiphyllum exhibits α-amylase and α-glucosidase inhibitory activity and enhances insulin release in vitro

Diabetes is one of the most prevalent chronic diseases globally. In this study, major polyphenols (17.35 ± 0.93–36.66 ± 2.01 mg/g) and minor fucoxanthin (non detected 15.12 ± 0.09 mg/g) were isolated from water, ethanol, and acetone extracts (WES, EES, and AES, respectively) of Sargassum hemiphyllum. Inhibition of α-amylase, α-glucosidase, sucrose, and maltase activities and stimulation of insulin secretion was greater with AES than with WES or EES and correlated with polyphenol and fucoxanthin concentrations in extracts. Moreover, 250 μg/ml EES and AES significantly increased insulin secretion in the presence of 25 mg/ml glibenclamide to higher levels than those obtained with 50 mg/ml glibenclamide. None of the extracts exhibited cytotoxicity, exacerbated the side effects of glibenclamide, or inhibited glibenclamide-induced insulin secretion. These results suggested that the S. hemiphyllum extracts WES, EES, and AES could be used as pharmaceuticals and functional foods to reduce dosages of synthetic diabetes drugs.