Name: semi-quantitative analysis of peptidoglycan by liquid chromatography mass spectrometry and bioinformatics
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Description: Watch this Scientific Journal Video about Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics at JoVE.com

作者要感谢Jennifer Geddes-McAlister博士和Anthony Clarke博士在完善该协议方面的贡献。这项工作得到了CIHR授予C.M.K（PJT 156111）的运营赠款和授予E.M.A.的NSERC Alexander Graham Bell CGS D的支持 BioRender.com。

1. 肽聚糖样品制备
<ol><li>细菌培养物的生长 注意：细菌培养物的生长将根据细菌种类和所检查的生长条件而有所不同。要测试的实验参数将定义生长条件。<ol><li>在细菌菌株和实验设计所需的生长条件下培养细菌培养物。以一式三份培养物（生物重复）的形式培养细菌，即每个菌株或生长条件三个单独的菌落。 注意：已知生长条件和生长阶段对PG组成1...

<ol>
	<li>Vollmer, W., Blanot, D., de Pedro, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan+structure+and+architecture.">Peptidoglycan structure and architecture.</a> FEMS Microbiology Reviews. 32, 149-167 (2007).</li><li>Pazos, M., Peters, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan.">Peptidoglycan.</a> Sub-cellular Biochemistry. 92, 127-168 (2019).</li><li>Typas, A., Banzhaf, M., Gross, C. A., Vollmer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+regulation+of+peptidoglycan+synthesis+to+bacterial+growth+and+morphology.">From the regulation of peptidoglycan synthesis to bacterial growth and morphology.</a> Nature Reviews. Microbiology. 10 (2), 123-136 (2011).</li><li>Yadav, A. K., Espaillat, A., Cava, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+strategies+to+preserve+cell+wall+integrity+against+environmental+threats.">Bacterial strategies to preserve cell wall integrity against environmental threats.</a> Frontiers in Microbiology. 9, 2064 (2018).</li><li>Bera, A., Herbert, S., Jakob, A., Vollmer, W., G&#246;tz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+are+pathogenic+staphylococci+so+lysozyme+resistant?+The+peptidoglycan+O-acetyltransferase+OatA+is+the+major+determinant+for+lysozyme+resistance+of+Staphylococcus+aureus.">Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus.</a> Molecular Microbiology. 55 (3), 778-787 (2005).</li><li>Brott, A. S., Clarke, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidoglycan+O-acetylation+as+a+virulence+factor:+its+effect+on+lysozyme+in+the+innate+immune+system.">Peptidoglycan O-acetylation as a virulence factor: its effect on lysozyme in the innate immune system.</a> Antibiotics. 8 (3), 94 (2019).</li><li>Putty, S., Vemula, H., Bobba, S., Gutheil, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+liquid+chromatography-tandem+mass+spectrometry+assay+for+D-Ala-D-Lac:+A+key+intermediate+for+vancomycin+resistance+in+vancomycin-resistant+Enterococci.">A liquid chromatography-tandem mass spectrometry assay for D-Ala-D-Lac: A key intermediate for vancomycin resistance in vancomycin-resistant Enterococci.</a> Analytical Biochemistry. 442 (2), 166-171 (2013).</li><li>Arthur, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+in+vivo+incorporation+of+D-lactate+into+peptidoglycan+precursors+of+vancomycin-resistant+Enterococci.">Evidence for in vivo incorporation of D-lactate into peptidoglycan precursors of vancomycin-resistant Enterococci.</a> Antimicrobial Agents and Chemotherapy. 36 (4), 867-869 (1992).</li><li>Mardarowicz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolierung+und+Charakterisierung+des+Murein-Sacculus+von+Brucella.">Isolierung und Charakterisierung des Murein-Sacculus von Brucella.</a> Z. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+for+the+enzymes+catalyzing+the+O-acetylation+of+bacterial+cell+wall+polysaccharides.">Assays for the enzymes catalyzing the O-acetylation of bacterial cell wall polysaccharides.</a> Methods in Molecular Biology. 1954, 115-136 (2019).</li><li>Clarke, A. 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该协议描述了一种从细菌培养物中纯化肽聚糖的方法，LC-MS检测过程并使用生物信息学技术分析成分。在这里，我们专注于革兰氏阴性细菌，需要进行一些轻微的修改才能分析革兰氏阳性细菌。
自 1960 年代首次生产以来，多肽的制备几乎保持不变9，11，15.纯化后，囊（第1.2.18节）使用来自球孢链<...

肽聚糖（PG）是细菌的一个决定性特征，用于维持细胞形态，同时为蛋白质和其他细胞成分提供结构支持1，2。PG的骨架由交替的β-1，4-连接的N-乙酰胞壁酸（MurNAc）和N-乙酰氨基葡萄糖（GlcNAc）1，2组成。每个MurNAc都具有一个结合在ᴅ-乳酰残基上的短肽，该肽可以与相邻的二糖连接的肽交联（图1A，B）。这种交联产生一种网状结构，包括整个细胞，通常被称为囊（图1C）。在PG合成过程中，前体在细胞质中产生，并通过翻转酶穿过细胞质膜。前体随后通过转糖基化酶和转肽酶掺入成熟的PG中，分别产生糖苷键和肽键3。然而，一旦组装，细菌产生的许多酶会修饰和/或降解PG以执行许多细胞过程，包括生长和分裂。此外，PG的各种修饰已被证明具有特异性的应变，生长条件和环境应激的适应性，这与细胞信号传导，抗菌素耐药性和宿主免疫逃避有关4。例如，一种常见的修饰是在MurNAc上添加C6乙酰基，通过限制聚糖β-1，4键与宿主产生的溶菌酶的访问来赋予抗性，从而降解PG4，5，6。在肠球菌中，用ᴅ-Lac取代肽侧链的末端ᴅ-Ala赋予抗菌剂万古霉素7，8更大的抗性。
PG分离和纯化的一般程序自1960年代9中描述以来一直保持相对不变。通过SDS热处理溶解细菌膜，然后酶促去除结合的蛋白质，糖脂和剩余的DNA。纯化的完整囊随后可以通过水解GlcNAc和MurNAc之间的β-1，4键消化成单个组分。这种消化产生具有任何结构修饰和/或交联完整的GlcNAc-MurNAc二糖，称为多肽（图1B）。
PG的成分分析最初通过高压液相色谱分离（HPLC）进行，以纯化每种多肽，然后手动鉴定多肽10，11。此后，液相色谱串联质谱（LC-MS）取代了液相色谱串联质谱（LC-MS），提高了检测灵敏度并减少了纯化每种多肽的手动工作量。然而，手动鉴定多肽的耗时性和复杂性仍然是一个限制因素，减少了进行的研究数量。近年来，随着“组学”技术的出现，自动化LC-MS特征提取已成为一种强大的工具，可以从非常大的数据集中快速检测和鉴定复杂样品中的单个化合物。一旦确定了特征，生物信息学软件就会使用差异分析统计比较样本之间的差异，从而隔离复杂数据集之间的最小差异，并以图形方式向用户显示。特征提取软件在PG组成分析中的应用才刚刚开始探索12，13，14，并与生物信息学分析相结合12。蛋白质组学分析得益于现成的蛋白质数据库，可预测肽片段化，可实现全自动鉴定，而蛋白质组学则不同，目前没有用于肽糖组学的片段化库。然而，特征提取可以与已知和预测的结构数据库相结合，以预测多肽鉴定12。在这里，我们提出了一个详细的方案，用于使用基于LC-MS的特征提取与多肽库相结合，对PG组成进行自动鉴定和生物信息差异分析（图2）。

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C18 reverse phase column - AdvanceBio Peptide column (100 mm x 2.1 mm 2.7 &#181;m)</td>
			<td>Agilent</td>
			<td></td>
			<td>LC-MS data acquisition</td>
		</tr>
		<tr>
			<td>Heating mantle controller, Optichem</td>
			<td>Fisher</td>
			<td>50-401-788</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>Heating Mantle, 1000mL Hemispherical</td>
			<td>Fisher</td>
			<td>CG1000008</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>Incubator, 37&#176;C</td>
			<td></td>
			<td></td>
			<td>for sacculi purification and MS sample prep</td>
		</tr>
		<tr>
			<td>Leibig condenser, 300MM 24/40,</td>
			<td>Fisher</td>
			<td>CG121805</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td></td>
			<td>for lyophilization of sacculi</td>
		</tr>
		<tr>
			<td>Magentic stirrer</td>
			<td>Fisher</td>
			<td>90-691-18</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>mass spectrometer Q-Tof model UHD 6530</td>
			<td>Aglient</td>
			<td></td>
			<td>LC-MS data acquisition</td>
		</tr>
		<tr>
			<td>microcentrifuge filters, Nanosep MF 0.2 &#181;m</td>
			<td>Fisher</td>
			<td>50-197-9573</td>
			<td>cleanup of sample before MS injection</td>
		</tr>
		<tr>
			<td>Retort stand</td>
			<td>Fisher</td>
			<td>12-000-102</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>Retort clamp</td>
			<td>Fisher</td>
			<td>S02629</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>round bottom flask - 1 liter pyrex</td>
			<td>Fisher</td>
			<td>07-250-084</td>
			<td>for 4% SDS boil</td>
		</tr>
		<tr>
			<td>Sonicator model 120</td>
			<td>Fisher</td>
			<td>FB120</td>
			<td>for sacculi purification</td>
		</tr>
		<tr>
			<td>Sonicator - micro tip</td>
			<td>Fisher</td>
			<td>FB4422</td>
			<td>for sacculi purification</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td></td>
			<td>sacculi wash steps</td>
		</tr>
		<tr>
			<td>Ultracentrifuge bottles, Ti45</td>
			<td>Fisher</td>
			<td>NC9691797</td>
			<td>sacculi wash steps</td>
		</tr>
		<tr>
			<td>Water supply</td>
			<td>City</td>
			<td></td>
			<td>for water cooled condenser</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemdraw</td>
			<td>Cambridgesoft</td>
			<td></td>
			<td>molecular editor for muropeptide fragmentation prediction</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>viewing lists of annotated muropeptides, abundance, isotopic patterns, etc.</td>
		</tr>
		<tr>
			<td>MassHunter Acquisition</td>
			<td>Aglient</td>
			<td></td>
			<td>running QTOF instrument</td>
		</tr>
		<tr>
			<td>MassHunter Mass Profiler Professional</td>
			<td>Aglient</td>
			<td></td>
			<td>bioinformatic differential analysis</td>
		</tr>
		<tr>
			<td>MassHunter Personal Compound Database and Library Manager</td>
			<td>Aglient</td>
			<td></td>
			<td>muropeptide m/z MS database</td>
		</tr>
		<tr>
			<td>MassHunter Profinder</td>
			<td>Aglient</td>
			<td></td>
			<td>recursive feature extraction</td>
		</tr>
		<tr>
			<td>MassHunter Qualitative analysis</td>
			<td>Aglient</td>
			<td></td>
			<td>viewing MS and MS/MS chromatograms</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td>Graphing software</td>
		</tr>
		<tr>
			<td>Perseus</td>
			<td>Max Plank Institute of Biochemistry</td>
			<td></td>
			<td>1D annotation</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher</td>
			<td>A998-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Fisher</td>
			<td>A637</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylase</td>
			<td>Sigma-Aldrich</td>
			<td>A6380</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Fisher</td>
			<td>BP168-1</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Fisher</td>
			<td>EN0521</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma-Aldrich</td>
			<td>27001-500ML-R</td>
			<td></td>
		</tr>
		<tr>
			<td>LC-MS tuning mix - HP0321</td>
			<td>Agilent</td>
			<td>G1969-85000</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Mutanolysin from Streptomyces globisporus ATCC 21553</td>
			<td>Sigma-Aldrich</td>
			<td>M9901</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen gas (&#62;99% purity)</td>
			<td>Praxair</td>
			<td>NI 5.0UH-T</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid</td>
			<td>Fisher</td>
			<td>A242</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase E from Streptomyces griseus</td>
			<td>Sigma-Aldrich</td>
			<td>P5147</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase</td>
			<td>Fisher</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Fisher</td>
			<td>S0489</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Sigma-Aldrich</td>
			<td>452890</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Fisher</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher</td>
			<td>S318</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (dibasic)</td>
			<td>Fisher</td>
			<td>S373</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (monobasic)</td>
			<td>Fisher</td>
			<td>S369</td>
			<td></td>
		</tr>
		<tr>
			<td>Stains-all</td>
			<td>Sigma-Aldrich</td>
			<td>E9379</td>
			<td></td>
		</tr>
	</tbody>
</table>

semi-quantitative analysis of peptidoglycan by liquid chromatography mass spectrometry and bioinformatics

肽聚糖是细菌细胞壁的重要组成部分，也是抗菌剂的常见细胞靶标。尽管肽聚糖结构的各个方面在所有细菌中都相当保守，但革兰氏阳性/阴性之间以及物种之间也存在相当大的差异。此外，肽聚糖有许多已知的变异、修饰或适应，这些变异、修改或适应可能发生在细菌物种内，以响应生长阶段和/或环境刺激。这些变异产生一种高度动态的结构，已知该结构参与许多细胞功能，包括生长/分裂、抗生素耐药性和宿主防御避免。为了了解肽聚糖内的变化，必须将整体结构分解为其组成部分（称为多肽）并评估整体细胞组成。肽糖组学使用先进的质谱法结合高功率生物信息学数据分析来详细检查肽聚糖组成。以下协议描述了从细菌培养物中纯化肽聚糖，通过液相色谱仪 - 质谱仪获取多肽强度数据，以及使用生物信息学对肽聚糖组成进行差异分析。

MS机器的检测灵敏度提高，加上高性能峰识别软件，提高了分离、监测和分析复杂样品物质成分的能力。利用这些技术进步，最近对肽聚糖组成的研究已开始使用自动化LC-MS特征提取技术12，13，14，24，而不是旧的基于HPLC的方法11，25，26，27，28，29，<sup c...

Watch this Scientific Journal Video about Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics at JoVE.com

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

该协议涵盖了使用液相色谱质谱结合高级特征提取和生物信息学分析软件对肽聚糖组成的详细分析。

肽聚糖的液相色谱质谱和生物信息学半定量分析

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Research

JoVE Journal

Biochemistry

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

A Tandem Liquid ChromatographyÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

单核苷酸延长和 MALDI 质谱分析校对及 DNA 修复方法

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

血管紧张蛋白II图解的硝酸分析与鉴定

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...