基质辅助激光解吸电离飞行时间质谱对阪崎肠杆菌的鉴定

目的 利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)法对阪崎肠杆菌进行鉴定,建立一种高效检测阪崎肠杆菌的方法,并为该技术的推广使用及阪崎肠杆菌的进一步研究提供科学依据.方法 用MALDI-TOF-MS法检测38株野生阪崎肠杆菌、2株标准菌株和1株阴沟肠杆菌,结果与常规生化鉴定结果对比;同时对在不同培养基上培养的阪崎肠杆菌进行质谱分析比较,对比不同培养基对质谱结果是否有影响;对38株野生菌株质谱图进行聚类分析.结果 38株菌株鉴定结果均为阪崎肠杆菌,与生化鉴定结果一致,且质谱鉴定分值大多在2.0以上.通过MALDI-TOF-MS鉴定方法可以很明显地将阴沟肠杆菌与阪崎肠杆菌两种菌分开.4种培养基对MALDI-TOF-MS鉴定结果的影响不是很明显,TSA比较适合作为阪崎肠杆菌MALDI-TOF-MS鉴定的培养基.通过质谱图谱和离子峰值比较得出,所有菌株在5745 m/z附近均出现高的离子峰,在2871、4740、8288、6260和9488 m/z附近出现离子峰的实验菌株达95％以上;在差异水平在0.5时,MALDI-TOF-MS的聚类分析结果可将所有实验菌株分成5个类型,结合菌株对应的来源和种类分析表明本研究所用菌株与来源和种类之间并无明显关系.结论 MALDI-TOF-MS方法具有准确且精确鉴定阪崎肠杆菌的能力;离子峰5745m/z具有作为阪崎肠杆菌的标记性离子峰的可能;差异水平为0.5进行MALDI-TOF-MS聚类分析,未发现5个类型与来源等具有一定关系,需要进一步研究.     

基质辅助激光解吸电离飞行时间质谱技术用于常见益生菌鉴定的初步研究

目的建立基质辅助激光解吸电离飞行时间质谱(MADLI-TOF MS)技术鉴定常见益生菌的实验方法并对MADLI-TOF MS技术的适用性进行初步评价。方法对MADLI-TOF MS技术鉴定常见益生菌过程中各影响因素进行考察,筛选出最佳的实验条件。利用19株供试菌株所得的蛋白指纹图谱对MADLI-TOF MS技术的适用性进行研究。结果建立了MADLI-TOF MS技术鉴定常见益生菌的最佳实验方法。初步证明MADLI-TOF MS技术具备在属、种、亚种以及菌株水平上鉴定常见益生菌的能力。结论建立的实验方法稳定性高、重复性好,可以作为MADLI-TOF MS技术鉴定常见益生菌的参考方法。MADLI-TOF MS技术可以作为常见益生菌鉴定的方法之一。     

基质辅助激光解吸电离飞行时间质谱技术作为常见益生菌菌种鉴定辅助工具的研究

目的评价基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术用于常见益生菌菌株鉴定及潜在益生菌菌株筛选的可行性。方法利用16S rDNA序列分析在方法学上对MALDI-TOF MS技术的鉴定能力进行研究;通过MALDI-TOF MS技术对现有保藏菌株的鉴定结果研究MALDI-TOF MS技术的鉴定准确性及优越性。结果 MALDI-TOF MS技术具备较16S rDNA序列分析更高的菌株鉴定能力;MALDI-TOF MS技术的鉴定结果准确、稳定。结论 MALDI-TOF MS技术可以作为准确、快速、廉价及可高通量操作的菌株鉴定方法应用于常见益生菌菌株的鉴定及潜在益生菌菌株的筛选。     

基质辅助激光解析电离飞行时间质谱在医学真菌领域的应用进展

基质辅助激光解析电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS）是近年来新兴的微生物检测技术,通过核糖体蛋白分析实现对真菌快速、准确鉴定。本文针对MALDI-TOF MS用于致病真菌鉴定、分类、体外抗真菌药物敏感性检测以及临床微生物样本直接检测等方面作一综述。     

应用基质辅助激光电离飞行时间质谱快速鉴定临床酵母菌

目的研究基质辅助激光解析电离飞行时间质谱(Matrix-Assisted Laser Desorption Ionization-Time of Flight MassSpectrometry,MALDI-TOF-MS)用于快速检测鉴定临床分离的酵母菌的可行性。方法应用Bruker MALDI-TOF-MS和VITEK 2-compact系统分别鉴定150株临床分离的酵母菌,结果不一致的菌株通过基因序列测定来鉴定。结果 MALDI-TOF-MS快速准确鉴定出了150株临床酵母菌,鉴定符合率在属水平上为100%,种水平上为94%。结论基于MALDI-TOF-MS鉴定方法具有很好的可重复性和准确性,并且其检测成本较低,实验准备时间很短,MALDI-TOF-MS可以用于临床分离的酵母菌的快速鉴定。     

基于核糖体蛋白质标志物及基质辅助激光解吸电离飞行时间质谱技术快速鉴定糖丝菌属放线菌

【目的】糖丝菌属(Saccharothrix)是一类丝状稀有放线菌,在生物医药、工业酶制剂和环境修复等领域展现出应用价值。本研究尝试建立以核糖体蛋白质为标志物,利用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization-time of flight mass spectrometry,MALDI-TOF MS)技术鉴定糖丝菌属放线菌的方法。【方法】检索基因组数据库,提取糖丝菌属测序菌株15种核糖体蛋白质的序列并计算理论分子量;通过分子量比对分析糖丝菌属不同菌种之间及其模式菌株与邻近属菌种模式菌株之间15种核糖体蛋白质的匹配度,提出鉴定至菌种及属的核糖体蛋白质匹配数标准;选取目标属和非目标属菌种进行MALDI-TOF MS测试和分析并修正鉴定标准。【结果】将待测菌株的MALDI-TOF质谱峰与糖丝菌属各菌种模式菌株的15种核糖体蛋白质分别匹配,通过最大匹配数、质谱峰强度模式及特征质谱峰鉴定至属或种。【结论】本研究建立了基于15种核糖体蛋白质标志物及MALDI-TOF MS技术鉴定糖丝菌属放线菌的方法,可用于定向筛选和快速鉴...     

基质辅助激光解析电离飞行时间质谱与细菌的快速鉴定

用传统的方法鉴别细菌往往需要较长的时间(≥48h)和复杂的程序,不利于细菌的快速鉴定。基质辅助激光解析电离飞行时间质谱是最近采用的用于细菌检测的生物质谱技术,可以对完整的细菌进行检测。这种技术以激光作为能量来源,将待测细菌的表面成分解析为离子,产生重现性很好的质谱图。将未知细菌质谱图与细菌质谱图库进行比较,可以达到对细菌进行鉴剐的目的。此种质谱技术的应用,对微生物的快速鉴别有重要意义。     

采用基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF)鉴定曲霉属Flavi组和Fumigati组的一些种（英文）

一些曲霉是主要的食物腐败菌及人的病原体,特别是免疫系统受损的病人可能导致严重的感染。本研究评价了利用基质辅助激光解吸电离飞行时间质谱对一些临床和环境中重要的Flavi组和Fumigati组曲霉,进行鉴定的可能性,并将结果与形态学及测序结果(ITS区和部分-tubulin和钙调蛋白基因)进行比较分析。通过曲霉中34个Flavi组菌株和30个Fumigati组菌株的质谱分析,来建立基质辅助激光解吸电离飞行时间质谱的数据库。对光谱数据进行聚类分析表明Fumigati组的基质辅助激光解吸电离飞行时间质谱的结果与系统发育结果完全一致;Flavi组的基质辅助激光解吸电离飞行时间质谱方法将A.flavus,A.oryzae,A.sojae和A.parasiticus分开的效果比测序方法好。随后,再选取用于验证数据库的50个菌株中49个(98%)菌株用质谱数据得到正确鉴定。对于分离本研究中曲霉的隐形种,基质辅助激光解吸电离飞行时间质谱方法优于测序方法。这种方法可以用于曲霉临床实验室鉴定的标准方法,因为临床需要快速和稳定的鉴定方法,这对于适当的治疗方案的选择很重要,此方法同样适于环境研究工作。     

基质辅助激光解吸附电离飞行时间串联质谱应用于液体培养细菌的鉴定

目前, 利用质谱直接鉴定细菌的方法基本是应用固体培养基获得的单克隆细菌, 该方法耗时较长. 因此建立基质辅助激光解吸附电离飞行时间串联质谱直接分析液体培养细菌, 通过正交实验, 以2, 5-二羟基苯甲酸(50 mg/mL, 50%乙腈-0.1%三氟乙酸)为基质获得最佳质谱图. 通过主成分分析能将不同种细菌区别开. 在混合培养时, 每种细菌的主要特征峰能在混合样品的质谱图中找到, 并且该方法灵敏度为1.8×10³. 结果表明, 该方法快速、灵敏, 可用于临床样本的快速辅助检测.     

MALDI-TOF-MS在病原微生物鉴定中的研究进展

基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)是鉴定多种致病性细菌的快速、可靠的方法,具有较好的稳定性和可重复性,在快速和准确性方面的总体表现明显好于传统的细菌生化鉴定方法。适合于一些致病菌的快速、高通量的检测和鉴定。综述MALDI-TOF-MS技术在普通病原菌、多血清型病原菌、非发酵性细菌,以及植物病原菌等病原微生物鉴定方面的最新研究进展。     

Metabolite profiling of plant carotenoids using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Although modern MS has facilitated the advent of metabolomics, some natural products such as carotenoids are not readily compatible to detection by MS. In the present article, we describe how matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) can be utilized to acquire mass spectra of carotenoids effectively. The procedure is sensitive (pmole range), reduces 'spot to spot' variation and provides high mass accuracy, thus aiding identification. The technique has been applied in vivo to the analysis of carotenoids in isolated plant cells and in vitro to three applications: (i) to show compatibility with purification methods such as LC, TLC and HPLC; (ii) for the rapid identification and quantification (by isotope dilution) of carotenoids present in crude extracts from plant tissues and whole cells; (iii) simultaneous semi-quantitative determination of carotenoids metabolites (m/z values) in crude plant extracts. Multivariate analysis of the recorded m/z values shows the effectiveness of the procedure in distinguishing genotypes from each other. In addition, the utility of the technique has been demonstrated on two mutant tomato populations, to determine alterations in carotenoid content, and a comparison made with traditional HPLC-photodiode array analysis. These data show that MALDI/TOF-MS can be used to rapidly profile, identify and quantify plant carotenoids reproducibly, as well as detecting other metabolites (m/z) in complex biological systems.     

MALDI-TOF MS技术在临床病原真菌鉴定中的应用进展

基质辅助激光解吸电离飞行时间质谱技术（MALDI-TOF-MS）目前是一种快速而可靠的微生物鉴定方法.随着可鉴定真菌谱的完善,MALDI-TOF MS技术已逐步应用于临床常见致病酵母菌、酵母样真菌和丝状菌的鉴定中,本文将就此做一综述.     

Capillary electrophoretic characterization of PEGylated human parathyroid hormone with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A capillary electrophoretic method (CE) for characterizing PEGylated human parathyroid hormone 1-34 (PTH) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described. CE was used to optimize the PEGylation of PTH through control of the reaction pH and the molar ratio of reactants with the advantages of minimal sample consumption and high separation capacity. The mono-PEGylated PTH (mono-PEG-PTH) was isolated and then digested with endoproteinase Lys-C. Resistance to Lys-C digestion on the PEGylation sites in the mono-PEG-PTH resulted in patterns of CE electropherograms different from that of the native PTH, and the PEGylation sites were assigned accordingly. The extent of positional isomers present in the mono-PEG-PTH was also determined by quantifying PEGylated fragments in the same CE electropherogram. In conclusion, the CE analysis of the Lys-C-digested sample allowed for simultaneous analysis of the PEGylation site and the extent of positional isomers in the mono-PEG-PTH. The results were confirmed by MALDI-TOF MS. This method will be applicable for characterizing PEGylation of other therapeutic peptides.     

Localization of water-soluble carbohydrates in wheat stems using imaging matrix-assisted laser desorption ionization mass spectrometry

The pool of endogenous water-soluble oligosaccharides found in the stems of wheat (Triticum aestivum) is being investigated as a potential indicator of grain yield. Techniques such as liquid chromatography with mass spectrometry (LC-MS) can profile these analytes but provide no spatial information regarding their distribution in the wheat stem. The imaging matrix-assisted laser desorption ionization (MALDI) mass spectrometry technique has not been utilized for the analysis of oligosaccharides in plant systems previously. Imaging MALDI mass spectrometry was used to analyse cross and longitudinal sections from the stems of Triticum aestivum. A range of oligosaccharides up to Hex(11) were observed. Water-soluble oligosaccharides were ionized as potassiated molecules, and found to be located in the stem pith that is retained predominantly around the inner stem wall. Imaging MALDI analyses provided spatial information on endogenous oligosaccharides present in wheat stems. The technique was found to offer comparable sensitivities for oligosaccharide detection to those of our established LC-MS method, and has potential for broad application in studying the in situ localization of other compound types in plant material.     

Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid identification of bacteria cultured in liquid media

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used for many years to rapidly identify whole bacteria. However, no consistent methodology exists for the rapid identification of bacteria cultured in liquid media. Thus, in this study we explored the use of MALDI-TOF MS analysis for rapid identification of cells cultured in liquid media. We determined that 2,5-dihydroxybenzoic acid (50 mg mL^?1, 50% acetonitrile, 0.1% trifluoroacetic acid) was the best matrix solution for MALDI-TOF MS for this type of study. Moreover, the tested strains were successfully differentiated by principal component analysis, and the main characteristics of the mass peaks for each species were found in mixed culture samples. In addition, we found that the minimum number of cells for detection was 1.8×10³. In conclusion, our findings suggest that MS-based techniques can be developed as an auxiliary method for rapidly and accurately identifying bacteria cultured in liquid media.     

A study on in vitro glycation processes by matrix-assisted laser desorption ionization mass spectrometry

The number of glucose molecules condensed on glycated bovine serum albumin have been easily determined by means of matrix-assisted laser desorption/ionization mass spectrometry. Measurements were carried out on samples from incubation of the proteins with glucose at different concentrations (0.02 M, 0.2 M, 2 M and 5 M). A clear increase in molecular mass of BSA with respect to incubation time is detected. In contrast to what is observed with fluorescence, the plots of molecular mass increase vs. incubation time show tha occurrence of a steady state, corresponding to the complete saturation of all the protein sites against glucose. Comparison of fluorescence and molecular mass data reveals that some further reactions, different from condensation, must take place, which could be in principle either intramolecular or originated by reactivity of modified condensed gluocse moieties vs. free glucose.