MicrobMatcher: a microbial comparison software based on matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry

Matrix-assisted Laser Desorption/Ionization with Time-of-flight Mass Spectrometry (MALDI-TOFMS) was investigated as a method for the rapid identifica-tion of species. Current demand in microbial identi-fication is how to compare unknown strains to the known one quickly, semi-automatically and accurately. In this paper, we present a software tool that allows flexibly microbial matching in a user-friendly way, by letting the users to customize comparison parameters including: in vitro transcription enzyme, mass tolerance,minimum fragment length, intensity threshold and corresponding weights. We provide three spectral scoring functions to compute the affin-ity between the species. Therefore, the precision of microbial comparison increases. To test and verify this tool, we employed experimental spectral data based on MALDI-TOFMS and the gene sequences of E.coli and Salmonella. This software is written in Java for cross-platform intention.

MicrobIdentifier: A Microbial Identification Software Based on Mass-Spectrometry

As the technology of microbial identification by mass cataloging has been widely used, we have developed the micro-bial identification software, MicrobIdentifier, which integrates and automates different steps in the procedure of rapid species identification based on mass-spectrometry. This software is written in Java for cross-platform intention.

Offline two-dimensional liquid chromatography coupled with ion mobility-quadrupole time-of-flight mass spectrometry enabling fourdimensional separation and characterization of the multicomponents from white ginseng and red ginseng

Inherent complexity of plant metabolites necessitates the use of multi-dimensional information to accomplish comprehensive profiling and confirmative identification.A dimension-enhanced strategy,by offline two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry(2 D-LC/IM-QTOF-MS)enabling four-dimensional separations(2 D-LC,IM,and MS),is proposed.In combination with in-house database-driven automated peak annotation,this strategy was utilized to characterize ginsenosides simultaneously from white ginseng(WG)and red ginseng(RG).An offline 2 DLC system configuring an Xbridge Amide column and an HSS T3 column showed orthogonality 0.76 in the resolution of ginsenosides.Ginsenoside analysis was performed by data-independent high-definition MSE(HDMSE)in the negative ESI mode on a Vion?IMS-QTOF hybrid high-resolution mass spectrometer,which could better resolve ginsenosides than MSEand directly give the CCS information.An in-house ginsenoside database recording 504 known ginsenosides and 58 reference compounds,was established to assist the identification of ginsenosides.Streamlined workflows,by applying UNIFI?to automatedly annotate the HDMSEdata,were proposed.We could separate and characterize 323 ginsenosides(including 286 from WG and 306 from RG),and 125 thereof may have not been isolated from the Panax genus.The established 2 D-LC/IM-QTOF-HDMSEapproach could also act as a magnifier to probe differentiated components between WG and RG.Compared with conventional approaches,this dimensionenhanced strategy could better resolve coeluting herbal components and more efficiently,more reliably identify the multicomponents,which,we believe,offers more possibilities for the systematic exposure and confirmative identification of plant metabolites.

Dimension-Enhanced Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry Combined with Intelligent Peak Annotation for the Rapid Characterization of the Multiple Components from Seeds of Descurainia sophia

The complex composition of herbal metabolites necessitates the development of powerful analytical techniques aimed to identify the bioactive components.The seeds of Descurainia sophia(SDS)are utilized in China as a cough and asthma relieving agent.Herein,a dimension-enhanced integral approach,by combining ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry(UHPLC/IMQTOF-MS)and intelligent peak annotation,was developed to rapidly characterize the multicomponents from SDS.Good chromatographic separation was achieved within 38 min on a UPLC CSH C18(2.1×100 mm,1.7μm)column which was eluted by 0.1%formic acid in water(water phase)and acetonitrile(organic phase).Collision-induced dissociation-MS^(2)data were acquired by the data-independent high-definition MS^(E)(HDMS^(E))in both the negative and positive electrospray ionization modes.A major components knockout strategy was applied to improve the characterization of those minor ingredients by enhancing the injection volume.Moreover,a self-built chemistry library was established,which could be matched by the UNIFI software enabling automatic peak annotation of the obtained HDMS^(E)data.As a result of applying the intelligent peak annotation workflows and further confirmation process,a total of 53 compounds were identified or tentatively characterized from the SDS,including 29 flavonoids,one uridine derivative,four glucosides,one lignin,one phenolic compound,and 17 others.Notably,four-dimensional information related to the structure(e.g.,retention time,collision cross section,MS^(1)and MS^(2)data)was obtained for each component by the developed integral approach,and the results would greatly benefit the quality control of SDS.

Studies on Fragmentation Regularity of 5-Methoxycarbonyl- 4-substituted-6-methyl-3,4-dihydropyrimidin-2(1H)-ones by Time-of-flight Mass Spectrometry

The electron impact time-of-flight(TOF) mass spectra of the title compounds were studied to establish their fragmentation processes. With the high resolution of the TOF instrument, the exact mass for each fragment was determined. These data were used to infer the molecular formulas and the elemental compositions for all the molecular ions and fragments through software interpretation. By further applying the fragmentation regularity, the majority of ions were fully assigned. The main fragmentation pathways of the title compounds include the formation of molecular ions by the loss of R 1 groups in the 4-position and the ester groups in the 5-position. The formed ion can be further fragmented by the elimination of MeOH.

The Development of Time-of-flight Mass Spectrometry( TOF-MS) and Its Applications in Screening Food Hazard Factors

The time-of-flight mass spectrometry(TOF-MS),one of the mass spectrometry techniques,has been widely applied in the field of rapid screening of food hazard factors as a superior analysis technique because of its wide mass range,high resolution and mass measurement accuracy,high sensitivity and high analysis speed.In this paper,research progresses of TOF-MS on the detection of food additives,food contaminants and residues,illegal additives,pesticide residues,veterinary drug residues and mycotoxin were reviewed,and its probable approaching applications were prospected.

Application of Liquid Chromatography-High Resolution Time-of-flight Mass Spectrometry in the Detection of Raw Milk and Dairy Products

Milk and dairy products are more and more popular with consumers due to their various nutrients, and their quality and safety issues have always been concerned. Therefore, the development of rapid, accurate and simple screening techniques is of great significance. Liquid chromatography-high resolution time-of-flight mass spectrometry has high-resolution and high-throughput detection functions, and has gradually begun to be applied in the detection of milk and dairy products. This paper summarized the application of milk and dairy products in liquid chromatography-high resolution time-of-flight mass spectrometry, laying a foundation for the development of new methods.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry traces the geographical source of Biomphalaria pfeifferi and Bulinus forskali,involved in schistosomiasis transmission

Background Freshwater snails of the genera Bulinus spp.,Biomphalaria spp.,and Oncomelania spp.are the main intermediate hosts of human and animal schistosomiasis.Identification of these snails has long been based on mor-phological and/or genomic criteria,which have their limitations.These limitations include a lack of precision for the morphological tool and cost and time for the DNA-based approach.Recently,Matrix-Assisted Laser Desorp-tion/lonization Time-Of-Flight(MALDI-TOF)mass spectrometry,a new tool used which is routinely in clinical microbi-ology,has emerged in the field of malacology for the identification of freshwater snails.This study aimed to evaluate the ability of MALDI-TOF MS to identify Biomphalaria pfeifferi and Bulinus forskali snail populations according to their geographicalorigin.Methods This study was conducted on 101 Bi.pfeifferi and 81 Bu.forskali snails collected in three distinct geo-graphical areas of Senegal(the North-East,South-East and central part of the country),and supplemented with wild and laboratory strains.Specimens which had previously been morphologically described were identified by MALDl-TOF MS[identification log score values(LSV)≥1.7],after an initial blind test using the pre-existing database.After DNA-based identification,new reference spectra of Bi.pfeiferi(n=10)and Bu.forskali(n=5)from the geographical areas were added to the MALDI-TOF spectral database.The final blind test against this updated database was per-formed to assess identification at the geographic source level.Results MALDI-TOF MS correctly identified 92.1%of 101 Bi.pfeifferi snails and 98.8%of 81 Bu.forskali snails.At the final blind test,88%of 166 specimens were correctly identified according to both their species and sampling site,with LSVs ranging from 1.74 to 2.70.The geographical source was adequately identified in 90.1%of 91 Bi.pfeifferi and 85.3%of 75 Bu.forskalii samples.Conclusions Our findings demonstrate that MALDI-TOF MS can identify and differentiate snail populations according to geographical origin.It outperforms the current DNA-based approaches in discriminating laboratory from wild strains.This inexpensive high-throughput approach is likely to further revolutionise epidemiological studies in areas which are endemic for schistosomiasis.

Improved preparation and identification of aristolochic acid-DNA adducts by solid-phase extraction with liquid chromatography-tandem mass spectrometry

Aristolochic acid （AA） is a known nephrotoxin and potential carcinogen, which can form covalent DNA adducts after metabolic activation in vivo and in vitro. A simple method for preparation and characterization of aristolochic acid-DNA adducts was developed. Four AA-adducts were synthesized by a direct reaction of AAI/AAII with 2′-deoxynucleosides. The reaction mixture was first cleaned-up and pre-concentrated using solid phase extraction （SPE）, and further purified by a reversed-phase high performance liquid chromatography （HPLC）. By the application of developed SPE procedure, matrices and byproducts in reaction mixture could be greatly reduced and adducts of high purity （more than 94% as indicated by HPLC） were obtained. The purified AA-DNA adducts were identified and characterized with liquid-electrospray ionization-quadrupole-time of flight-mass spectrometry （LC-ESI-Q-TOF-MS/MS） and LC-Diode array detector-fluorescence （LC-DAD-FL） analysis. This work provides a robust tool for possible large-scale preparation of AA-DNA adduct standards, which can promote the further studies on carcinogenic and mutagenic mechanism of aristolochic acids.

Characterization of 4-Aryl-5-ethoxycarbonyl-6-methyl-3,4-dihydro-pyrimidin-2(1H)-thiones by EI-TOF Mass Spectrometry

The electron impact （EI） time-of-flight mass spectra （TOFMS） of the title compounds （ thioxo-Biginelli compounds） were studied to establish their fragmentation pathways. With a TOF instrument of high resolution, the exact mass for each fragment was determined. These data were used to infer the molecular formulas and the ionic elemental compositions for all the compounds through interpretation by software. By further applying chemical intuition, majority of the fragmentation ions were fully assigned. All the compounds give strong signals（ average 76% ） for their molecular ions in the El spectra. Two kinds of characteristic fragmentation pathways from the molecular ion were observed. One is related to the loss of the ester group, forming a resonance stabilized ion with a moderate abundance（ average 40% ）. The other concerns the loss of the aromatic-ring radical, forming another resonance stabilized ion at m/z 199 with a high abundance（ average 79% ）, from which further important fragmentations including the formation of an ion at m/z 171 （average 24% ） by the loss of C2H2 （via the McLafferty rearrangement）, and an ion at m/z 153（ average 10% ） by the elimination of a water molecule preceed. In addition, ion [ M - Et ]^＋ with a high abundance（average 62% ） is a characteristic for the Biginelli compounds with an ethoxycarbonyl group. Differences and similarities among the fragmentations observed from the EI-TOFMS of oxo-Biginelli compounds are discussed.

Screening and Identifying of Nephrotoxic Compounds in Lithospermum erythrorhizon Using Live-cell Fluorescence Imaging and Liquid Chromatography Coupled with Tandem Mass Spectrometry

In order to identify the potential nephrotoxic compounds in traditional Chinese medicine Lithospermum erythrorhizon,it was separated into serial fractions according to their polarities.An in vitro method was utilized to determine the nephrotoxicity of these fractions with the help of fluorescence image analysis.As a result,the primary fraction A05 and its secondary fractions C06 ＂C09 and C12 ＂C14 were found to have significant toxicity to LLC-PK1 cell line,as determined by the survive rate less than 20% after they were treated with these fractions.These potential nephrotoxic fractions were further analyzed by multistage and high resolution mass spectrometry.The main compounds in these fractions were tentatively identified to be acetylshikonin,isobutyrylshikonin,β,β＇-dimethyla-cryloylshikonin,and isovalerylshikonin,which may bring nephrotoxicity.

淮安地区市售熟制坚果籽类产品和冲调谷物制品霉菌污染状况

目的了解淮安地区市售熟制坚果籽类产品和冲调谷物制品中霉菌污染状况,并比较18S rRNA测序和微生物飞行时间质谱两种方法在霉菌种属鉴定中的一致性。方法2022—2023年在淮安地区采集熟制坚果籽类产品和冲调谷物制品进行霉菌检测,并对检出的霉菌利用18S rRNA测序和微生物飞行时间质谱方法进行种属鉴定,比较两种方法的一致性。对检出率最高的青霉菌属进行进化分析,用MEGA 7.0软件构建进化树。结果检测样品179份,26份检出霉菌,检出率为14.5%。熟制坚果籽类产品霉菌检出率13.2%(10/76),1份霉菌超标;冲调谷物制品霉菌检出率15.5%(16/103)。26株霉菌经18S rRNA测序,种类为青霉菌属9株,曲霉菌属7株,枝孢霉属5株,交链孢属2株,小孢根霉、烟草赤星病菌、玫瑰黄链霉菌各1株。微生物飞行时间质谱共鉴定出14株,在属水平上与18S rRNA测序鉴定结果完全一致,种水平鉴定结果一致率为64.3%。9株青霉菌属进化分析显示,9株主要分为两大支,来自坚果和来自谷物制品的青霉菌属在同一分支,不同年份的菌株也会在同一分支上。结论淮安地区市售熟制坚果籽类产品和冲调谷物制品中霉菌检出率较高,应加强监管和风险评估。

不同碳氮比下后置反硝化工艺中溶解性有机质的分子特征及其转化机理

污水处理厂后置反硝化工艺中外加碳源的投加量会影响出水溶解性有机质(DOM)的特性,从而影响后置反硝化的处理效率和出水水质.本研究以实际污水中DOM为研究对象,利用傅里叶变换离子回旋共振质谱(FT ICR-MS)开展了不同碳氮比(C/N=3、4、5、6)下后置反硝化工艺中DOM的分子特征及其转化机理研究.结果表明,在4种C/N下,C/N=5的反应器出水芳香性DOM分子(AImod>0.5)和不饱和程度高的分子(DBE≥4)占比最高,分别为24.72%和97.6%,说明C/N=5时,出水难生物降解程度较高.通过分子转化分析可知,C/N=5的反应器产生的DOM分子占比(58.35%)最高且不饱和程度较高,去除的分子占比(2.80%)低且不饱和程度较低,再次证实C/N=5的反应器出水相较于C/N=3、4、6的反应器出水DOM分子惰性高,排放至受纳水体环境风险较低.此外,微生物群落结构及其与DOM的关联分析可知,Proteobacteria在C/N=5的反应器中相对丰度最高,其主导后置反硝化工艺中DOM分子的产生,Planctomycetota、Nitrospirota、Chloroflexi和Gemmatimonadota对DOM分子的去除和产生也具有重要作用.综上所述,推荐C/N比为5作为后置反硝化工艺中外加碳源的适宜C/N比.

乳酸菌发酵前后山药汁营养功能成分及风味物质的变化

研究了乳酸菌发酵对山药汁营养功能成分及感官特性的影响。结果显示,经L.plantarum 101和L.paracasei HGD组合发酵后的山药汁在总体感官上更容易被接受。与未发酵山药汁相比,乳酸菌发酵可将pH值由6.12降至3.88,总糖含量降低69.35%,总酚、可溶性膳食纤维、总酸和游离氨基酸的含量分别提高98.00%、53.15%、493.51%和64.30%。同时气相色谱-质谱分析结果表明,乳酸菌发酵可使山药汁中风味物质的种类及含量显著变化,其中酸类提高195.02%,酯类提高172.73%,酯类物质种类及含量的增加赋予了发酵山药汁丰富的香味;酮类和醇类分别提高88.27%和7.46%,醛类下降了85.82%,使得最终山药汁发酵液的可接受度提高。综上所述,山药汁经乳酸菌发酵后不仅能够提高其营养成分,还可以改善风味,该研究结果表明乳酸菌发酵山药汁可作为一种待开发的新型功能性饮品。

基于SPME-GC-MS分析稻谷遭受4种霉菌浸染期间挥发性物质成分的变化

对接种4种不同霉菌的稻谷样品中挥发性物质组分分析,探究其变化规律及特征性挥发性物质。首先,将4株霉菌(灰绿曲霉3.0100、灰绿曲霉3.3975、尖孢镰刀菌PM、尖孢镰刀菌PP)制成2种含量的菌悬液,再将其分别接种在稻谷样品上,于28℃、80%相对湿度的恒温培养箱中储藏56 d,每隔7 d用固相微萃取-气质联用技术(SPME-GC-MS)对稻谷样品进行检测,获得定性和定量数据,并进行分析。结果显示接种不同霉菌的稻谷样品在储藏期间产生的挥发性物质主要有烷、烯、醇、酮、醛、酸、酯、芳香族化合物及其他类九大类物质,在储藏不同时间后,产生的挥发性物质种类与含量有较大差异,从中找出了4种霉变稻谷的特征性挥发性物质和在不同污染阶段的变化规律。初步研究表明,通过检测稻谷中的挥发性物质来判断污染霉菌的种类和生长状态的有一定的可行性。

中国东北干旱-半干旱地区湖泊沉积物木质素酚类化合物特征及其气候指示意义

木质素广泛分布于维管植物,经分解生成的酚类化合物可示踪有机质来源、评估木质素降解程度,进而用于反演古环境与古气候变化。采用合适的分析方法有效地分解木质素是推断母源植物类型、降解程度的技术基础,常规方法是木质素经碱(或酸)解后,利用气相色谱-质谱法(GC-MS)分析酚类单体化合物,但分解、提取过程复杂、易引入杂质。热裂解技术可在高温下快速分解有机质,裂解产物可通过GC-MS进行在线分析,具有用样量少、有机质提取比例高、重现性好、操作便捷的特点。本文选择地处亚洲夏季风影响区域的边缘的内蒙伊和沙日乌苏湖,采用热裂解GC-MS(Py-GC/MS)技术,对湖泊沉积物进行裂解分析,在对裂解温度(450℃、550℃和650℃)进行了优化的基础上,识别了21种酚类化合物,包括:4-甲基苯酚、2-乙基苯酚等9种烷基酚类(PHs),4-乙基-2-甲氧基苯酚、4-乙烯基-2,6-二甲氧基苯酚等9种烷基酚类(PHs)和12种甲氧基酚类(LGs)。结合沉积岩心样品AMS 14 C年龄的分析结果,6.7 ka以来沉积物中酚类化合物总量、PHs和LGs的变化趋势总体一致,呈现出6.7~4.0ka相对含量较高、4.0ka以来相对含量较低的特征。不同于PHs中邻(o-)-PHs、间(m-)-PHs、对(p-)-PHs的变化趋势与总量一致;但不同取代特征的LGs相对含量变化趋势存在差异,p-LGs在5.4ka前后就出现含量显著下降,3.8ka以来维持较低水平。根据微生物对木质素的“去甲基/去甲氧基”氧化反应途径,对位取代酚类化合物比值(p-PHs/p-LGs)可作为陆生高等植物降解指标,该值越大微生物降解作用越强。将p-PHs/p-LGs指标应用于伊和沙日乌苏沉积物样品结果显示,6.7ka以来p-PHs/p-LGs与正构烷烃单体碳同位素δ^(13) C_(27-33)变化趋势一致(R=-0.77),间接地指示了有效降水变化。即6.7ka以来气候整体转湿,区内陆生高等植物占据优势,充足的水分和有机质为微生物提供了适宜的生存环境和相对稳定的营养来源,降解作用整体呈增强趋势;6.3~5.5ka和4.1~3.6ka期间有效湿度降低,微生物对木质素的降解作用相对减弱。p-PHs/p-LGs指标对应了呼伦贝尔地区湿度变化特征,揭示了干旱-半干旱地区微生物降解与有效湿度变化的相关性,为探讨陆地生态系统对东亚季风北部边缘区气候变化的响应提供科学依据。

基于高通量液相色谱质谱技术的菌株筛选与关键分子定量分析研究进展

合成生物学是21世纪新兴的交叉学科,已经为医药、化工、能源、食品和农业等领域带来了显著的改变。微生物细胞工厂(microbial cell factory,MCF)的构建和应用是合成生物学的重要研究内容,正迅速向实用化、产业化的方向发展。得益于基因编辑技术的进步,MCF菌株文库构建的能力大幅提升,亦产生了数量庞大的待测样本,亟需发展高通量自动化的分析检测方法与之匹配。此外,针对关键代谢酶或代谢物的高通量检测技术,也对MCF改造乃至工业化生产有重要的指导意义。液相色谱质谱技术是生物分子检测分析的重要手段,在蛋白质、代谢物定量分析方面占据重要地位。因此本文总结回顾了近年来高通量液相色谱质谱技术在微生物菌株筛选及关键分子定量分析方面的研究进展,从样品制备、色谱分离、质谱分析及数据处理等层面展开阐述,并对这一领域与自动化平台结合的前景及如何深度融合的挑战进行简要概括,为推动基于合成生物学生物智造领域的快速发展提供指导。

3种气单胞菌鉴定方法的比较

采用分子生物学检测方法、API 20NE生化鉴定法以及梅里埃(VITEK MS)全自动快速微生物质谱检测法,对60株试验菌株进行种属鉴定,比较3种鉴定方法的准确性和优缺点。对试验菌株进行16S r DNA特异性引物扩增和测序,确定菌株种属,并作为比较3种鉴定方法的标准。随后用API 20NE生化鉴定法和梅里埃(VITEK MS)全自动快速微生物质谱检测法,对试验菌株进行鉴定。结果显示,分子生物学方法和生化鉴定法,对60株菌的检出率均为100%,而质谱仪的检出率仅为60.0%。使用分子生物学法共检出44株气单胞菌(100%),准确率为100%;使用生化鉴定法对气单胞菌的检出率为88.6%,准确率为59.1%;使用质谱仪对气单胞菌的检出率为54.5%,准确率为38.6%。3种鉴定方法对气单胞菌的检出率和准确率从大到小为:分子生物学法、API 20NE生化鉴定、质谱仪。

原位电离质谱技术在微生物菌株筛选中的应用进展

质谱是一种强大的分析工具,可提供分子量和化学结构信息。它具有高特异性、高灵敏度、快速、普适性、微量和非标记等优点。在合成生物学的“设计-构建-测试-学习”工程化策略中,质谱具有重要的应用潜力。随着质谱仪器及其方法体系的不断发展,质谱,特别是原位电离质谱技术,已经成为检测和筛选微生物菌株的重要工具。它能够获取完整的细胞代谢表型,用于合成生物学中的“测试”环节高通量筛选和成像。本文重点介绍了经典的基质辅助激光解吸/电离质谱技术和基于电喷雾、激光和等离子体的原位电离质谱技术的工作机制。此外,还综述了这些质谱技术可以在无需样品预处理的情况下直接检测完整的微生物细胞,以及在微生物突变文库的高通量筛选和活微生物菌落的质谱成像方面的研究进展。最后,总结了原位电离质谱技术在合成生物学中的应用。原位电离质谱技术具有微量、无标记、高通量、普适性、高灵敏度等特点,将在合成生物学“测试”环节的高通量筛选装备中发挥重要作用。