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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>&#945;-cyano-4-hydroxy-cinnamic acid</td>
			<td>ACROS Organics</td>
			<td align="right">163440050</td>
			<td>&#8805; 97%, CAS 28168-41-8</td>
		</tr>
		<tr>
			<td>MALDI calibration kit</td>
			<td>Aigma-Aldrich</td>
			<td>MSCAL1-1KT</td>
			<td>This kit also contains acetonitrile, trifluoroacetic acid , sinapinic acid, etc.</td>
		</tr>
		<tr>
			<td>MALDI target plate</td>
			<td>Bruker Daltonics</td>
			<td align="right">280800</td>
			<td>Polished Steel</td>
		</tr>
		<tr>
			<td>Bruker Microflex LRF MALDI-TOF mass spectrometer</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></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...

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

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 ...

科研

生物学

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

酰胺氢/氘交换和MALDI - TOF质谱PAK2激活的质谱分析

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

利用数字图像相关表征的血管组织标本本地菌株

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

完整的工作流程为组蛋白翻译后修饰的分析采用自下而上质谱法：从组蛋白提取数据分析

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. Research on such captive populations can also yield valuable data to address key information gaps about the behavior, life history, and ecology of the target taxa. We describe a protocol for captive propagation of the federally endangered Cyclargus thomasi bethunebakeri that can be used as a model for other at-risk butterfly ex situ programs, especially those in the family Lycaenidae. We further provide a simple and straightforward protocol for recording various life history metrics that can be useful for informing ex situ methodologies as well as adapted for laboratory studies of other lepidoptera.

Here, we present protocols for 1) the laboratory captive propagation of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri), and 2) assessing basic life history information such as immature development time and number of larval stadia. Both methods can be adapted for use with other ex situ conservation programs.

风险蝴蝶捕获繁殖计划，以提高生命历史知识和有效的异地保护技术

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.