基质辅助激光解吸飞行时间质谱及毛细管电泳对基因工程产品─重组人表皮生长因子的质量控制研究

本文应用基质辅助激光解吸飞行时间质谱（ＭＡＬＤＩ－ＴＯＦ－ＭＳ）测定基因工程产品一重组人表皮生长因子（ｒｈＥＧＦ）的分子量，用毛细管电泳仪测定了其纯度，并得到用Ｇｌｕ－蛋白酶切后ｒｈＥＧＦ的质量肽图，其中８１％的肽段与理论值相符，无规则裂解只占８％。由此建立了一种对基因工程产品进行质量控制及研究蛋白质一级结构的新方法，此方法快速、简便，灵敏度极高，相对误差小于０．５％。     

Aerobic biodegradation of polyethylene glycols of different molecular weights in wastewater and seawater

In order to distinguish between aerobic biodegradation of synthetic polymers in fresh and seawater, polyethylene glycols (PEGs) were systematically and comparatively investigated in inocula from municipal wastewater and seawater aquarium filters for the first time. The molecular weight (MW) of the PEGs, (HO(CH(2)CH(2)O)(n)H, n=3-1350) as representatives of water-soluble polymers, ranged from 250 to 57,800Da. The biodegradation was observed by removal of dissolved organic carbon and carbon dioxide production by applying standardized ISO and OECD test methods. Specific analyses using liquid chromatography mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were performed. All PEGs selected were completely biodegradable in freshwater media within 65d. PEGs with an MW up to 14,600Da have a similar degradation pathway which is characterized by gradual splitting of C(2)-units off the chain resulting in formation of short-chain PEGs. In artificial seawater media, full biodegradation of PEGs up to 7400Da required more time than in freshwater. PEGs with MW 10,300 and 14,600Da were only partially degraded whereas PEGs with MW 26,600 and 57,800Da were not degraded for a period of 135d. The biodegradation pathway of PEG 250 and PEG 970 in seawater is similar to that for freshwater. For PEGs having an MW from 2000 to 10,300Da, the degradation pathway in seawater differs from the pathway of the shorter PEGs.     

Sulphated polysaccharides from Indian samples of Enteromorpha compressa (Ulvales,Chlorophyta): Isolation and structural features

Heteroglycan and xyloglucan rich fractions were extracted from Indian samples of Enteromorpha compressa in 25% yield by sequential extractions with water and alkali. This heteroglycan is sulfated and has an apparent molecular mass of 55 kDa. Chemical structural analysis of this polysaccharide revealed a branched structure having 1,4- and 1,2,4-linked rhamnose 3-sulphate, 1,4-linked glucose, 1,3- and 1,6- linked galactose, 1,4- and terminally linked glucuronic acid and 1,4-linked xylose partially sulfated on O-2. Chemical and spectroscopic analysis showed that the 4-M KOH extracted hemicellulosic fraction contained an unusual β-(1,4)-linked linear xyloglucan. Enzyme hydrolysis and analysis of the resulting fragments by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) showed that this linear polymer contained partially sulfated Glc₃Xyl₂ or Glc₄Xyl₂ as oligomeric building subunits.     

蜡样芽孢杆菌在草莓中的生长及预测模型的建立

在对草莓的病源微生物污染调查中发现蜡样芽孢杆菌具有高的检出率。为比较不同温度和不同包装方式的草莓表面蜡样芽孢杆菌数量的变化情况,测定了7℃、15℃、25℃、30℃的温度条件下,采用保鲜盒包装和保鲜膜包装方式的草莓表面蜡样芽孢杆菌生长数据;选用Baranyi Roberts模型为初级模型,拟合不同温度和不同包装方式的草莓中蜡样芽孢杆菌生长曲线,并对模型进行验证。结果显示,7℃冷藏的蜡样芽孢杆菌均增长缓慢而与包装方式无关;15℃低温贮藏过程中,膜装与盒装延滞期基本相同,但膜装比盒装降低了0.7 lg cfu/g的生长势,说明保鲜膜在此温度下有一定的抑菌作用;在25℃、30℃储藏下两种包装方式的比生长速率和延滞期基本相同,蜡样芽孢杆菌在42 h后分别增长了2.9 lg cfu/g、3.2 lg cfu/g,表明贮藏温度是影响蜡样芽孢杆菌的主要因素。因此在高温条件下蜡样芽孢杆菌会带来潜在的食用安全风险。本研究结果为微生物定量风险评估提供重要信息。     

霍乱弧菌基质辅助激光解吸飞行时间质谱数据库的建立与应用

目的建立基质辅助激光解吸电离飞行时间质谱法(matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF-MS)快速检测霍乱弧菌,并建立数据库。方法考察培养基、培养时间、培养温度以及样品前处理方法对图谱质量和鉴定结果的影响,对方法的稳定性、重复性进行研究;采集101株霍乱弧菌食品分离株MALDI-TOF-MS指纹图谱,并对图谱进行分析。其中标准菌株CICC23794及40株分离株作为建库菌株,其余60株用于建库后的测试。结果采用2%NaCl-TSA培养基,在37℃、24h条件下培养,可达到强峰值信号,谱图重现性良好,甲酸-乙腈提取法获得的质谱图特征峰多,信噪比高;建库后, 60株霍乱弧菌均可达到高于2.0的水平,其中约有20株霍乱弧菌鉴定分值可达到2.4以上。结论本研究建立的霍乱弧菌MALDI-TOF-MS鉴定方法结果准确,可用于实际检测。     

克罗诺杆菌MALDI-TOF-MS数据库的建立及应用

应用基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption ionisation time-of-flight mass spectrometry,MALDI-TOF-MS）技术建立了克罗诺杆菌MALDI-TOF-MS数据库,用于该菌属内种和亚种的高通 量鉴定及菌株分型。在优化培养条件、样品处理方法及确定MALDI-TOF-MS蛋白质指纹图谱采集参数的基础上, 将采集的8 种参考菌株蛋白质质谱数据通过Biotyper软件构建克罗诺杆菌的MALDI-TOF-MS数据库,再用相近肠杆 菌及该属分离株验证数据库的准确性。结果表明：应用建立的克罗诺杆菌的MALDI-TOF-MS数据库对135 株克罗 诺杆菌分离株进行分析,鉴定分值均不小于2.0,达到了种水平鉴定要求,通过聚类分析,可进一步分型。构建的 MALDI-TOF-MS数据库为克罗诺杆菌高通量鉴定与分型提供了一种新的手段。     

单增李斯特氏菌MALDI-TOF-MS鉴定与分型研究

为建立单增李斯特氏菌的基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)快速鉴定与分型方法,实验收集37株单增李斯特氏菌分离株,应用MALDI-TOF-MS采集图谱,获取独特的蛋白质指纹图谱,汇总成标准图谱,建立单增李斯特氏菌鉴定数据库。采用单增李斯特氏菌标准菌株进行验证,表明鉴定结果的可信度很高。在数据库信息的基础上,对37株单增李斯特氏菌分离株进行聚类分型。分型结果表明,在蛋白质水平上,MALDI-TOF-MS可把37株单增李斯特氏菌分成9个型别。     

MALDI-TOF mass spectrometry detection of extra-virgin olive oil adulteration with hazelnut oil by analysis of phospholipids using an ionic liquid as matrix and extraction solvent

The adulteration of extra virgin olive oil (EVOO) with hazelnut oil (HO) is frequent and constitutes a serious concern both for oil suppliers and consumers. The high degree of similarity between the two oils as regards triacylglycerol, total sterol and fatty acid profile, complicates the detection of low percentages of HO in EVOO. However, phospholipids (PLs) are usually present in seed oils at a concentration range of 10–20 g/kg, while the amounts of PLs in VOOs are 300–400 times lower. Thus, in this work a sample pretreatment procedure focused towards the selective PLs extraction was developed; the Bligh–Dyer extraction procedure was modified introducing the ionic liquid resulting from the combination of TBA (tributylamine) and CHCA (α-Cyano-4-hydroxycinnamic acid) as extraction solvent. The selective extraction and enrichment of phospholipids from EVOO and HO samples was then achieved. The relevant extracts were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using the same ionic liquid TBA-CHCA as MALDI matrix, that was found to be very suitable for PLs analysis. In fact, a remarkable increase of the phospholipids signals, with a simultaneous decrease of those relevant to triacylglycerols and diacylglycerols, was observed in the relevant mass spectra. The applicability of the whole method to the individuation of the presence of HO in EVOO was demonstrated by the analysis of EVOO samples progressively adulterated with variable quantities of HO, that was still detectable at a 1% contamination level.     

MALDI-TOF-MS分析技术及在聚合物表征中的应用

基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)作为一种高效、精确的分析方法,已经在生物、化学、生命科学等很多领域得到了广泛应用。本文着重阐述了MALDI-TOF-MS的构造、性能特征,以及在聚合物分析中的应用。