肽聚糖的液相色谱质谱和生物信息学半定量分析
Summary
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。
Abstract
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。
Introduction
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）。
Protocol
1. 肽聚糖样品制备 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。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…
Representative Results
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…
Discussion
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<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Equipment | |||
| C18 reverse phase column – AdvanceBio Peptide column (100 mm x 2.1 mm 2.7 µm) | Agilent | LC-MS data acquisition | |
| Heating mantle controller, Optichem | Fisher | 50-401-788 | for 4% SDS boil |
| Heating Mantle, 1000mL Hemispherical | Fisher | CG1000008 | for 4% SDS boil |
| Incubator, 37°C | for sacculi purification and MS sample prep | ||
| Leibig condenser, 300MM 24/40, | Fisher | CG121805 | for 4% SDS boil |
| Lyophilizer | Labconco | for lyophilization of sacculi | |
| Magentic stirrer | Fisher | 90-691-18 | for 4% SDS boil |
| mass spectrometer Q-Tof model UHD 6530 | Aglient | LC-MS data acquisition | |
| microcentrifuge filters, Nanosep MF 0.2 µm | Fisher | 50-197-9573 | cleanup of sample before MS injection |
| Retort stand | Fisher | 12-000-102 | for 4% SDS boil |
| Retort clamp | Fisher | S02629 | for 4% SDS boil |
| round bottom flask – 1 liter pyrex | Fisher | 07-250-084 | for 4% SDS boil |
| Sonicator model 120 | Fisher | FB120 | for sacculi purification |
| Sonicator – micro tip | Fisher | FB4422 | for sacculi purification |
| Ultracentrifuge | Beckman | sacculi wash steps | |
| Ultracentrifuge bottles, Ti45 | Fisher | NC9691797 | sacculi wash steps |
| Water supply | City | for water cooled condenser | |
| Software | |||
| Chemdraw | Cambridgesoft | molecular editor for muropeptide fragmentation prediction | |
| Excel | Microsoft | viewing lists of annotated muropeptides, abundance, isotopic patterns, etc. | |
| MassHunter Acquisition | Aglient | running QTOF instrument | |
| MassHunter Mass Profiler Professional | Aglient | bioinformatic differential analysis | |
| MassHunter Personal Compound Database and Library Manager | Aglient | muropeptide m/z MS database | |
| MassHunter Profinder | Aglient | recursive feature extraction | |
| MassHunter Qualitative analysis | Aglient | viewing MS and MS/MS chromatograms | |
| Prism | Graphpad | Graphing software | |
| Perseus | Max Plank Institute of Biochemistry | 1D annotation | |
| Material | |||
| Acetonitrile | Fisher | A998-4 | |
| Ammonium acetate | Fisher | A637 | |
| Amylase | Sigma-Aldrich | A6380 | |
| Boric acid | Fisher | BP168-1 | |
| DNase | Fisher | EN0521 | |
| Formic acid | Sigma-Aldrich | 27001-500ML-R | |
| LC-MS tuning mix – HP0321 | Agilent | G1969-85000 | |
| Magnesium chloride | Sigma-Aldrich | M8266 | |
| Magnesium sulfate | Sigma-Aldrich | M7506 | |
| Mutanolysin from Streptomyces globisporus ATCC 21553 | Sigma-Aldrich | M9901 | |
| Nitrogen gas (>99% purity) | Praxair | NI 5.0UH-T | |
| Phosphoric acid | Fisher | A242 | |
| Pronase E from Streptomyces griseus | Sigma-Aldrich | P5147 | |
| RNase | Fisher | EN0531 | |
| Sodium azide | Fisher | S0489 | |
| Sodium borohydride | Sigma-Aldrich | 452890 | |
| Sodium dodecyl sulfate (SDS) | Fisher | BP166 | |
| Sodium hydroxide | Fisher | S318 | |
| Sodium Phosphate (dibasic) | Fisher | S373 | |
| Sodium Phosphate (monobasic) | Fisher | S369 | |
| Stains-all | Sigma-Aldrich | E9379 |
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Cite This Article
Anderson, E. M., Greenwood, N. A., Brewer, D., Khursigara, C. M. Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics. J. Vis. Exp. (164), e61799, doi:10.3791/61799 (2020).