Amidation of methyl-esterified oligogalacturonides: examination of the reaction products using MALDI-TOF MS

Amidation of methyl-esterified oligogalacturonides (oligoGalA) was studied to produce partly and fully amidated oligoGalA to be used as substrates and/or inhibitors for the characterization of pectolytic enzymes acting on the homogalacturonan backbone. The reactions were performed with varying concentrations of ammonia or methylamine and monitored in time using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that allows sensitive monitoring of the reactions. MALDI-TOF MS reveals the degree of amidation (DAm) and extent of hydrolysis of methyl-esters. Using this technique the conditions for each of the reactions was optimized. Amidation was performed best under anhydrous conditions at a concentration of 4 M ammonia or methylamine at ambient temperature. Amidation using methylamine reached almost completeness (DAm 95) without hardly any hydrolysis of methyl-esters while amidation with ammonia reached a DAm of 70 on average. After an initial fast amidation, precipitation of the partly amidated oligoGalA reduced the reaction rate enormously. The use of ammonia in aqueous solutions instead off anhydrous ammonia resulted in 6–10% lower DAm values due to the hydrolysis of methyl-esters. Therefore, anhydrous conditions are preferred during amidation. Furthermore, methylamine is a better reagent for amidation of oligoGalA and pectins then ammonia, but also results in totally different products with other properties.     

Global changes in phospholipids identified by MALDI MS in rats with focal cerebral ischemia

Neuronal membrane phospholipids are highly affected by oxidative stress caused by ischemic injury. Thus, it is necessary to identify key lipid components that show changes during ischemia to develop an effective approach to prevent brain damage from ischemic injury. The recent development of MALDI imaging MS (MALDI IMS) makes it possible to identify phospholipids that change between damaged and normal regions directly from tissues. In this study, we conducted IMS on rat brains damaged by ischemic injury and detected various phospholipids that showed unique distributions between normal and damaged areas of the brain. Among them, we confirmed changes in phospholipids such as lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin by MALDI IMS followed by MS/MS analysis. These lipids were present in high concentrations in the brain and are important for maintenance of cellular structure as well as production of second messengers for cellular signal transduction. Our results emphasize the identification of phospholipid markers for ischemic injury and successfully identified several distinctly located phospholipids in ischemic brain tissue.     

基质辅助激光解吸电离飞行时间质谱在微生物鉴定中的研究进展

与传统的微生物鉴定技术相比,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)是一种准确、可靠和快速的鉴定和分型的技术。本文通过检索近年来国内外相关研究论文,总结最新的研究进展,发现MALDI-TOF MS在临床病原微生物、食源性微生物以及环境微生物等鉴定中有较大的优势,加快了微生物鉴定的进程,同时探索该技术在新领域的最新进展和面临的挑战,以期为我国基质辅助激光解吸电离飞行时间质谱技术的发展提供参考。     

Domain swapping in p13suc1 results in formation of native-like, cytotoxic aggregates

The field of protein aggregation has been occupied mainly with the study of beta-strand self-association that occurs as a result of misfolding and leads to the formation of toxic protein aggregates and amyloid fibers. However, some of these aggregates retain native-like structural and enzymatic properties suggesting mechanisms other than beta-strand assembly. p13suc1 is a small protein that can exist as a monomer or a domain-swapped dimer. Here, we show that, under native conditions, p13suc1 forms three-dimensional domain-swapped aggregates, and that these aggregates are cytotoxic. Thus, toxicity of protein aggregates is not only associated with beta-rich assemblies and amyloid fibers, involving non-native interactions, but it can be induced by oligomeric misassembly that maintains predominantly native-like interactions.     

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。     

六种猪呼吸道RNA病毒MALDI-TOF MS同步检测体系的构建

【目的】建立猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)、猪瘟病毒(classical swine fever virus,CSFV)、口蹄疫病毒(foot-and-mouth disease virus,FMDV)和猪流感病毒(swine influenza virus,SIV)的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)多目标检测方法,同时对SIV进行通用型、H1型及H3型分型检测。【方法】本研究根据6种病原体基因的保守序列,设计了6对加标引物及对应的延伸探针并进行单反应试验。通过体系优化引物浓度和反应条件,以及方法特异性、重复性及灵敏度分析,使用MALDI-TOF MS检测方法及荧光定量PCR方法分别对临床样本和猪源产制品进行检测,并对结果进行对比验证。【结果】质谱结果显示,6种产物峰仅在靶标病毒对应的产物位置出现峰...     

基于基质辅助激光解吸/电离飞行时间质谱的猪呼吸道病毒多目标鉴定方法的建立和应用

【目的】建立能高效同步鉴定猪伪狂犬病毒(porcine pseudorabies virus,PRV)、猪圆环病毒2型(porcine circovirus 2,PCV-2)和3型(porcine circovirus 3,PCV-3)、非洲猪瘟病毒(African swine fever virus,ASFV)以及猪博卡病毒1型(porcine bocavirus group 1,PBoV-G1)、2型(porcine bocavirus group 2,PBoV-G2)和3型(porcine bocavirus group 3,PBoV-G3)等呼吸道病毒的核酸基质辅助激光解吸/电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS)高通量多目标检测技术。【方法】根据7种病原体基因的保守序列,分别设计不同病原的引物及对应的单碱基延伸探针,通过引物浓度和反应条件优化,方法特异性、敏感性和稳定性分析,以及临床样本和猪源产制品的检测验证,建立常见猪呼吸道DNA病毒的MALDI-TOF MS多目标检测体系。【结果】质谱分析显示,多目标检测体系的7种靶标产物峰只在特定病毒阳性样品检测时产生,与其他病原体检测无交叉反应,表明该方法对7种靶标病毒检测特异性良好。重复性试验结果分析显示,体系中每种病毒在高、中、低浓度时批内阳性符合率均≥98.0%,批间均≥98.3%,表明该方法具有较高的稳定性。体系中7种病原体每种病毒最低检测限在8.65–26.27拷贝/μL之间,与荧光PCR(real-time fluorescence quantitative PCR,RT-qPCR)检测方法相当。采用MALDI-TOF MS多重检测方法对100份组织、饲料和猪肉样品进行检测应用,检出2种及以上混合感染样品39份,其中5份样本同步检出5种病原体阳性;对8份ASFV-p72假病毒人工污染样品进行验证,均可检出ASFV阳性。将以上样本检测应用结果与荧光PCR方法进行比对验证,2种方法对于不同病原体检测结果的符合率高达94.4%–100%。【结论】本研究建立的基于MALDI-TOF MS的猪呼吸道常见DNA病毒多重检测方法为猪群相关疫病快速监测和鉴别诊断,以及便利化进出口动物检疫等提供了一种新的敏感、特异的高通量多目标检测技术。     

蛋白质芯片SELDI-TOFMS技术的研究进展及其在临床中的应用

蛋白质芯片为新一代的蛋白质组研究技术,由美国Ciphergen生物系统公司引进,表面增强激光解吸电离-飞行时间质谱(SELDI-TOFMS)提供一个高通量和高灵敏度的检测平台。投放至今虽短短10来年,但卓越的成果已广为医学科学界重视,尤其在恶性肿瘤的早期诊断、监控和预后研究上。蛋白质是细胞内执行生物功能的最终分子,蛋白质组学研究让人类更深入了解疾病和生命的本源,不断发现的特异性肿瘤标志物更为攻克癌症带来新希望。这里除对表面增强激光解吸电离_飞行时间质谱作较详尽的介绍外,更重点阐述其近年来蛋白质芯片近期的研究进展和在临床中的应用,并就其优劣和发展前景作出评估。     

Variation between strains of the cyanobacterium Microcystis aeruginosa isolated from a Portuguese river

AIMS: The aim of this study was to investigate toxicological differences between strains of the cyanobacterium Microcystis aeruginosa isolated from a potable water supply in the north of Portugal over a 2-month period. METHODS AND RESULTS: Twenty-six strains of M. aeruginosa were isolated, grown in pure culture, and tested using a range of techniques including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), ELISA and a PCR procedure targeting the genes implicated in the production of toxic microcystins. There was considerable variation with respect to the amounts of microcystin produced by each of the strains as measured by ELISA, with values ranging from 0.02 to 0.53% dry weight. The results of the MALDI-TOF MS analysis demonstrated the presence of several chemically distinct forms of microcystin as well as aeruginosins, anabaenopeptins and several other unidentified peptide-like compounds. CONCLUSIONS: The growth of individual strains that comprise bloom populations, with unique 'chemotypes' can potentially be an important factor affecting the toxicity of bloom populations. Molecular probes, targeting the genes responsible for microcystin production were shown to be useful for distinguishing between toxic and nontoxic strains and showed good agreement with the results obtained from the other analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that the analysis of cyanobacterial bloom populations at the subspecies (strain) level can potentially provide important information regarding the toxin-producing potential of a cyanobacterial bloom and could be used as an 'early warning' for toxic bloom development.     

ITS序列分析与MALDI-TOF MS质谱技术在丝状真菌鉴定中的应用

丝状真菌常用的鉴定方法为形态方法和基因鉴定方法,前者限于检验人员的知识和技能,后者操作繁琐,费用略昂贵,不适合常规开展。因此,寻找丝状真菌快速鉴定方法势在必行。本文采用VITEK MALDI-TOF MS（基质辅助激光解析电离时间飞行质谱）IVD数据库（3.0版本）对临床分离的254株丝状真菌进行鉴定,并以ITS（internal transcribed spacer 内转录间隔区）序列分析为标准,验证MALDI-TOF MS质谱技术鉴定丝状真菌的准确性。结果表明MALDI-TOF MS质谱技术可以对大部分丝状真菌实现快速、准确的鉴定,其中对毛癣菌属（100%）、毛孢子菌属（100%）、毛霉菌属（100%）、曲霉菌属（96.5%）准确率很高,对犬小孢子菌（75%）、镰刀菌属（50%）、新月弯孢霉（46.2%）准确率较低,对丝状真菌鉴定的总体准确率为86.36%,与ITS测序分析符合率为83.97%。     

微生物学研究中的软电离质谱技术

包括基质辅助激光解吸电离（ＭＡＬＤＩ）和电喷雾（ＥＳＩ）在内的软电离质谱是最近发展起来的质谱技术,由于这些电离方式对样品的破坏性小,质量测定范围大,分子量测定准确,样品纯度要求不高很适合分析成分复杂的微生物样品,ＭＡＬＩＤＩ－ＴＯＦ－ＭＳ结合高分辨率的二维ＳＤＳ－ＰＡＧＥ可以分析１０＾－１２摩尔水平的蛋白,是细菌蛋白质研究过程中必不可少的工具。最近的研究工作表明,通过ＭＡＩＤＩ－ＴＯＦ－ＭＳ或ＨＰ     

Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.     

Chemical derivatization of phosphoserine and phosphothreonine containing peptides to increase sensitivity for MALDI-based analysis and for selectivity of MS/MS analysis

Protein phosphorylation is one of the most important and common ways of regulating protein function in cells. However, phosphopeptides are difficult to analyse, ionising poorly under standard MALDI conditions. Several methods have been developed to deal with the low sensitivity and specificity of phosphopeptide analysis. Here, we show an approach using a simple one-step beta-elimination/Michael addition reaction for the derivatization of phosphoserine and phosphothreonine. The substitution of the negatively charged phosphate group by a positively charged S-ethylpyridyl group greatly improves the ionisation of the modified peptides, especially in MALDI MS, increasing the sensitivity of the analysis. The modification allows the formation of a unique fragment ion at m/z 106 under mild collisional activation conditions, which can be used for parent (precursor) ion scanning in order to improve both the sensitivity and the selectivity of the analysis. The optimisation of the approach is described for a standard model peptide and protein and then applied to phosphorylation analysis in two biologically derived proteins purified from different experimental systems.     

Combination of Micropreparative Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Peptide Analysis

Signal suppression is a problem in matrix-assisted laser desorption/ionization mass spectrometry of peptides prepared by capillary electrophoresis. Many common electrolytes that are efficient for separation, such as sodium phosphate, also are strongly suppressive during laser desorption/ionization. We have tested individual electrolytes for highest performance in each step of separation and collection, respectively. Suppression is not observed if citrate, trifluoroacetic acid, or hydrochloric acid is used for collection, while phosphate still can be employed in the capillary providing excellent resolution. Low concentrations of hydrochloric acid added to the sample/matrix mixture generate mass spectra with better ion intensities than if trifluoroacetic acid or citrate is used.     

一种SDS-PAGE与MALDI-TOF质谱联用的方法

以尿激酶的为材料,探索一种从ＳＤＳ－ＰＡＧＥ胶上回收蛋白 做ＭＡＬＤＩ－ＴＯＦ质谱的方法,所用的回收方法包括电洗脱、脱盐、除ＳＤＳ等过程,电洗脱用的是高盐阻断法,脱盐用的超滤技术,去除ＳＤＳ用的是冷丙沉淀法,结果证明,此方法至少对一些蛋白质是可行的。     

一种SDS-PAGE与MALDI-TOF质谱联用的方法

以尿激酶原为材料,探索一种从SDS-PAGE胶上回收蛋白质做MALDI-TOF质谱的方法.所用的回收方法包括电洗脱、脱盐、除SDS等过程.电洗脱用的是高盐阻断法,脱盐用的是超滤技术,去除SDS用的是冷丙酮沉淀法.结果证明,此方法至少对一些蛋白质（如尿激酶原和牛血清白蛋白）是可行的.