Name: use of maldi-tof mass spectrometry and a custom database to characterize bacteria indigenous to a unique cave environment (kartchner caverns, az, usa)
Uploaded: 2015-01-02T15:00:00.000+00:00
Duration: 11 min 9 s
Description: Watch this Scientific Journal Video about Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA at JoVE.c

This work was supported by the New College of Interdisciplinary Arts and Sciences at Arizona State University, Applied Maths NV, and by the National Science Foundation (ROA Supplement to Award No. MCB0604300). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

注意 :在任何环境不明的细菌可能是致病的，必须使用适当的生物安全协议，谨慎处理。与活的文化工作，必须在II类生物安全柜使用生物安全2级（BSL-2）的程序进行。有关BSL-2程序的更多信息，请在CDC / NIH手册中标题为"生物安全微生物和生物医学实验室，"33-38页。该文件可在网上<a href="http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf">http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf</a> 。适当的个人防护装备（PPE），包括实验室大衣/袍，护目镜和腈或乳胶手套，必须佩戴。标准的微生物实践和注意事项必须遵循的，和生物危险废物，必须进行适当的丢弃。 在这个演示中使用细菌分离卡切内岩洞，AZ，USA，从4的环境中，包括干钟乳石，流石，湿润钟乳石和钟乳石滴（ 表1）。所有分离物的16S rDNA序列鉴定，并保持在-80℃下在25％的甘油，R 2 B培养基。所有实验均在室温下完成。 注:我们推荐使用相同的样品制备方法获得大规模光谱数据库建设和未知的质谱。样品制备方法以前已证明影响光谱质量和再现性3。使用不同的样品制备方法可能会导致不正确的识别未知的，特别是当更高分类分辨率（ 例如 ，在应变水平）是期望的。 1.沉积在MALDI靶 注意 :几个协议，以获得蛋白质提取物需要使用的酸和有机溶剂必须按照GUID被利用包含在各自的材料安全数据表（MSDS）elines和信息。合适的PPE必须佩戴并且将异基于化学品的使用的类型和量（ 例如 ，实验室外套/服，手套，安全眼镜，和呼吸保护必须用显著量的有毒的，易燃的溶剂，如乙腈中工作时使用的，和腐蚀性酸，如甲酸和三氟乙酸）。 <ol><li>存1微升不含存活细胞到不锈钢MALDI靶盘，并允许它以干燥（使用适当的，先前所描述的协议11-13得到的）的蛋白质提取物。覆盖干燥的蛋白质提取物与1μl的基质溶液（α氰基-4-羟基肉桂酸溶液），并允许其干燥。 </li><li>对于每一个生物重复，临场技术重复（5〜20技术复制）的适当数量。在这里，发现10技术复制的每个生物重复和3个生物学重复瓦特ERE准备。 注意:我们建议使用蛋白提取的样品制备方法时，使用抛光不锈钢MALDI靶板。使用地面钢靶板可能引起扩散和不同样品的无意混合单个样品井之外。 </li><li>存1微升校准标准到目标板，并让其干燥。覆盖用1微升基质溶液，并允许它干燥。 </li><li>存2微升基质溶液到目标板作为阴性对照。 </li></ol> 2.质谱采集<ol><li>使用MALDI-TOF质谱仪装有氮激光器（λ= 337纳米），并使用布鲁克FlexControl软件操作。 </li><li>由500激光投100拍摄的增量积累收集每个质谱正线性模式。设置在离子源1电压20千伏;离子源2的电压为18.15 kV的;和透镜电压，以9.05千伏。需要注意的是，这些参数是乐器特定FIC，可能需要其他手段的调整，以获得最佳的效果。 </li><li>从每次充电2至20 kDa的设定批量荷范围自动频谱评估。使用质心的峰值检测算法。定在100大的最小分辨率阈值。在2阈值100设定的最小强度阈值:将信号噪声比（N S）。 </li></ol> 3.数据库建设<ol><li>数据库设计<ol><li>创建BioNumerics 7.1一个新的数据库使用"新建数据库向导"。 </li><li>创建谱的实验类型， 例如，的Maldi，在"实验类型"面板中使用的命令。 </li><li>创建使用"数据库设计面板"的水平。使用添加了新的水平，在"数据库"菜单中的"等级&gt;添加新的水平..."命令。在这里，打造"种"的水平，"生物重复"水平"和"技术复制"的水平，respectiv伊利。 </li></ol></li><li>导入和预处理原质谱<ol><li>导出原始质谱为使用FlexAnalysis在"文件"菜单中单击"导出&gt;质谱"命令.txt文件。 </li><li>导入原料质谱（.txt文件）转换为数据库中的技术重复的水平。 </li><li>预处理原料质谱。 <ol><li>导入和重采样（使用二次拟合算法）。 </li><li>执行基线减法（与滚动光盘50点的尺寸）。 </li><li>计算噪音[连续小波变换（CWT），流畅（Kaiser窗以20分的窗口大小和10点测试），并执行第二基线减法（滚动光盘200点大小）。 </li><li>检测峰[CWT具有最小的信噪比（S:N）10]。 </li></ol></li><li>预处理后，保存每个质谱的特征模式，例如含峰峰列表大小，峰强度，S:N 等 ，在数据库中。 </li></ol></li><li>创建复合质谱<ol><li>创建从"分析"菜单中的"总结..."命令预处理光谱复合光谱。选择"生物重复"的目标水平。 </li><li>这里，结合同殖民地的10技术复制质谱产生复合质谱为殖民地，从而导致该菌株在"生物重复"三级复合质谱。 </li><li>在这里，总结一下三复合光谱创建一个复合谱是孤立的"种"的水平。 注意:合成频谱是逐点平均的技术重复的。具有相似性（Pearson相关），以低于95％（缺省设置）的平均复制被排除在复合材料。上的复合光谱峰仅称为如果它们存在于75％（在包括重复的默认设置）。对于生物学重复，这些设置分别为90和60％，分别为。 </li></ol></li></ol> 4.质谱数据分析<ol><li>选择在数据库中的条目，并创建由"比较"面板中单击"创建新的比较"命令比较。 </li><li>这里，可使用质谱在"技术复制"和/或"生物复制"的水平，显示的比较和分析。 </li><li>基于相似性的聚类分析和多维标度（MDS）的<ol><li>创建颜色组。选择三个生物复合质谱，然后单击"创建新组的选择"命令，在"组"菜单中创建一个组对应的分离。指定自动用于这三种质谱一种颜色。 </li><li>另外，定义域指出使用的命令，相应的颜色"数据库条目"面板，使得在此基础上定义的字段中的任何分组使用该组中定义的相同的颜色。 </li><li>进行聚类分析。在"群集"菜单中单击"计算聚类分析"命令。在比较设置页1，选择"Pearson相关"，离开其他参数为默认值。在第2页，选择"UPGMA"。然后单击"完成"。 </li><li>获得"统计"菜单中的MDS阴谋使用"多维尺度..."命令。 </li></ol></li><li>峰匹配<ol><li>在"实验"面板点击谱型"的Maldi"。然后选择"布局&gt;显示图像"。光谱被示为凝胶带。 </li><li>请在"光谱"菜单中的"做峰匹配"命令峰匹配。 </li></ol></li><li>峰值类鉴定<ol><li>执行主成分分析（PCA）。突出"实验MALDI"在实验型"，"面板，并使用"主成分分析..."命令，在"统计"菜单中执行PCA。 </li><li>执行双向聚类。点击"比较"窗口中的"统计&gt;矩阵挖掘"......。的匹配峰值类的峰的强度使用不同的颜色（热图）表示。 </li></ol></li></ol> 5，细菌鉴定与自定义数据库<ol><li>相似系数为基础的方法<ol><li>创建一个比较和按步骤4.3.3描述生成基于质谱在"技术复制"级树状图。保存树状的相似性比较。 </li><li>选择一个未知的质谱，并单击"分析&gt;确定入选作品"。出现在鉴定对话框。 </li><li>选择"比较基础"分类型（或存储classifIER），然后点击"下一步"。在接下来的页面中，选择保存的树状图作为参考比较，然后单击"下一步"。 </li><li>选择"基本相似性"作为一种识别方法，然后单击"下一步"。 </li><li>选择"最大的相似性"的评分方法。键入适当的阈值和最低值的差值为每个参数，然后单击"下一步"。 </li><li>一旦计算完成，将出现识别窗口。在"结果"面板中，数据库的成员最符合未知列。 </li><li>保存该项目鉴定和验证使用"交叉验证分析"命令识别，在"识别项目"面板。 </li></ol></li><li>潜在的生物标志物为基础的方法<ol><li>定义峰值类。在"黑客帝国矿业"窗口中，选择组峰具有共同的特点，并确定这些峰作为特定使用FIC峰值类（潜在生物标志物）"谱&gt;管理高峰类的类型......"中的"比较窗口"。 </li><li>在这里，定义特定的每个隔离所有15株峰值类。 </li><li>选择未知数的质谱和匹配这些光谱的峰到峰定义的类，如前所述。 </li></ol></li></ol>

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Authors Vranckx and Janssens are employees of Applied Maths NV, the manufacturer of data analysis software used in this video. Applied Maths NV provided select software modules highlighted in this video as well as a portion of the publication costs associated with this video.

该演示展示了表征和鉴定用MALDI-TOF MS和一个自定义数据库细菌的详细过程。相较于传统的分子生物学方法，例如，16S rDNA序列分析，MALDI-TOF MS的基于指纹的方法促进更快速鉴定多样的细菌。因为它的耐用性，这种技术被广泛用于从环境和临床环境1,14-16表征细菌，病毒，真菌和酵母。此外，MALDI-TOF MS已经报道，得到，在一些情况下，更高分类分辨率1。例如，B。藻。 A，B，D和...

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been shown to be a rapid and reliable tool for identification of bacteria at the genus, species, and in some cases, strain levels1-4. MALDI-TOF MS ionizes biological molecules (typically proteins) that originate from cell surfaces, intracellular membranes, and ribosomes from bacterial whole cells or protein extracts1,5. The resulting peaks form characteristic patterns or &#8220;fingerprints&#8221; of the bacteria analyzed1. Identification of bacteria is based on these mass-to-charge &#8220;fingerprints&#8221;. 
Two of the most commonly used identification strategies are library-based and bioinformatics-based strategies1. Library-based approaches involve comparing the mass spectra of unknowns to previously collected mass spectra of known bacteria in databases/libraries for identification. Commercially available software, such as BioNumerics, Biotyper, and SARAMIS software packages, as well as open source software tools, such as SpectraBank6, are available to facilitate the comparison and quantification of similarity between mass spectra of unknowns and reference bacteria. Bioinformatics-based approaches usually rely on fully sequenced genomes of bacteria for identification. In contrast to library-based approaches which do not involve identification of the biological nature of particular peaks, bioinformatics-based approaches involve protein identification1. 
The majority of recent MALDI fingerprint-based studies have used library-based approaches to identify bacteria1. Library-based approaches require construction of databases and comparison of the similarity between mass spectra. Studies show that many experimental procedures, such as medium3,7, cultivation time8, sample preparation method3, and matrix used9, affect the mass spectra obtained. Furthermore, some closely-related species and strains generate spectra with only subtle differences. Thus, library-based approaches require rigorously standardized procedures to generate highly reproducible mass spectra between replicates. Minor variations in protocols may compromise the efficacy of identification, especially at the subspecies and strain levels1,3,10. However, neither manufacturer-provided reference databases nor reported custom databases include visually documented procedures for database construction and/or application of a data analysis pipeline. For this reason, the objective of this work was to develop, apply, and demonstrate a comprehensive and detailed procedure for library-based bacterial identification using MALDI-TOF MS. 
In this demonstration, mass spectra of 15 bacteria isolated from a karstic environment (Kartchner Cavern, AZ, USA) were collected and imported into software to construct a model database. Data processing and the analysis pipeline were detailed using the model database. Finally, mass spectra of blind-coded bacteria which were randomly selected from these 15 bacteria were collected again and compared to the reference spectra in the model database for identification. Results show that bacteria can be correctly identified either based on similarity coefficients or potential biomarkers/peak classes.

<table><tbody><tr><td>&amp;alpha;-cyano-4-hydroxy-cinnamic acid</td><td>ACROS Organics</td><td>163440050</td><td>&amp;ge; 97%, CAS 28168-41-8</td></tr><tr><td>Bruker FlexControl software</td><td>Bruker Daltonics</td><td></td><td>version 3.0</td></tr><tr><td>Bruker FlexAnalysis software</td><td>Bruker Daltonics</td><td></td><td>version 3.0</td></tr><tr><td>Bionumerics software</td><td>Applied Maths</td><td></td><td>version 7.1</td></tr></tbody></table>

use of maldi-tof mass spectrometry and a custom database to characterize bacteria indigenous to a unique cave environment (kartchner caverns, az, usa)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

在此演示构建的数据库有四个层次，从最高到最低的水平，其中包括"各级"，"物种"，"生物重复"和"技术复制"，分别为（ 图1A）。 "技术复制"级别包含的技术复制所有的预处理光谱。在"生物重复"和"物种"的水平包含复合（摘要）光谱。 "各级"包含了所有的技术重复谱以及所有的复合光谱。 频谱聚合过程使用的是有代表性的峰， 如图1所示。每个成员?...

Watch this Scientific Journal Video about Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA at JoVE.c

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

use-maldi-tof-mass-spectrometry-custom-database-to-characterize

Research

JoVE Journal

Biology

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

13.9K 次观看.  Arizona State University. This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented. 

视频: Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms.
Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations.
The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP

Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST).

器的子类型<em&gt;空肠弯曲菌</em&gt; SSP。<em&gt; doylei</em&gt;分离株使用基于光谱质谱PhyloProteomics（MSPP）

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. ...

科研

遗传学

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

生物学

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

水生微生物Metaproteomics工作流程:从细胞对胰蛋白酶肽适合串联质谱为基础的分析

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and ...

环境科学

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

生物化学

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

免疫与感染

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定 大肠杆菌 中的抗菌免疫蛋白

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

化学

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

Conjunctival Commensal Isolation and Identification in Mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

小鼠的共性共性隔离和识别

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

MALDI-TOF Mass Spectrometry

Mass spectrometry is a powerful characterization technique that can identify and separate a wide variety of compounds ranging from chemical to biological entities, based on their mass-to-charge ratio (m/z). The instruments that allow this detection, known as mass spectrometers, have three components: an ion source, a mass analyzer, and a detector. These spectrometers differ based on the nature of their ion source and analyzers.
Matrix-assisted laser desorption ionization (MALDI) is a commonly used mass spectrometer. It enables the soft ionization of biological molecules like peptides, lipids, and saccharides without fragmenting them. This means they can be analyzed intact as the sample integrity is maintained.
In MALDI, the sample is mixed with a compatible matrix that is organic molecules. When the laser strikes the sample, the matrix functions as a mediator for energy absorption, yielding the intact sample analyte ions. The matrix molecules energetically ablate from the sample surface, absorb the laser energy and carry the sample molecules into the gas phase. The sample molecules are usually ionized during the ablation process to carry a single positive charge.
The desorbed sample ions carrying the charge are then directed towards the mass analyzer, usually a time-of-flight (TOF). The ions pass through a field-free drift region which allows separation based on size, allowing the smaller ions to reach the detector first.
MALDI-TOF instruments are often connected with a reflectron that reflects ions increasing the ion flight path. This increased flight time between ions of different m/z allows better sample resolution where ions of the same mass reach the detector simultaneously. MALDI sources can be coupled with other analyzers, including triple quadrupoles and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. The MALDI-FT-ICR is known for its high mass resolution, wherein the sample ions carrying the charge move to an ICR cell and cyclotron in a magnetic field while separating on m/z.
With a couple of limitations, like, the possibility of photodegradation by the laser for some sample types, absorption of the laser by a few fluorescent analytes that could affect the results, an acidic matrix that could alter sample nature, etc., overall, this technique has vast applications.&#160; It can analyze protein samples isolated by electrophoresis or chromatography, peptides, carbohydrates, DNA, identify microorganisms from clinical samples and ascertain biomolecules in tissues, among many others.

MALDI-TOF 质谱仪

Mass spectrometry is a powerful characterization technique that can identify and separate a wide variety of compounds ranging from chemical to biological ...

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

摘要

探索更多视频

此视频中的章节

器的子类型

MALDI样品制备：超薄层的方法

水生微生物Metaproteomics工作流程:从细胞对胰蛋白酶肽适合串联质谱为基础的分析

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定大肠杆菌中的抗菌免疫蛋白

通过成像质谱分析研究感染期间琼脂和组织中生长的细菌菌落的微生物合作

小鼠的共性共性隔离和识别

MALDI-TOF 质谱仪

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

摘要

探索更多视频

此视频中的章节

器的子类型

MALDI样品制备：超薄层的方法

水生微生物Metaproteomics工作流程:从细胞对胰蛋白酶肽适合串联质谱为基础的分析

使用开源 MALDI TOF-MS IDBac 管道分析微生物蛋白和专用代谢物数据

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定 大肠杆菌 中的抗菌免疫蛋白

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

小鼠的共性共性隔离和识别

MALDI-TOF 质谱仪

使用MALDI-TOF-TOF-MS/MS和自上而下的蛋白质组学分析鉴定大肠杆菌中的抗菌免疫蛋白

通过成像质谱分析研究感染期间琼脂和组织中生长的细菌菌落的微生物合作