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
Summary
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.
Abstract
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.
Introduction
微生物是无处不在，发挥在地球的生物地球化学循环1重要作用。目前，可用于表征微生物群落结构和功能众多的分子生物学方法。最常见的是16S rRNA基因的序列分析PCR扩增从环境的DNA 2 – 4。的16S rRNA基因分析的缺点在于，它仅提供了有关系统发育特征和社区结构信息，具有信息很少代谢功能。与此相反，方法，如宏基因组学，metatranscriptomics和metaproteomics提供群落结构和新陈代谢的信息。宏基因组学，或生物体的集合体的基因含量的分析，提供了有关社区5的结构和功能的潜在的信息– 8。虽然功能强大，该功能的潜在可能与代谢生物体的活动。一个生物体的基因型是由它的基因，其每一个可以被转录为RNA和进一步翻译为蛋白质，从而导致表型来表示。因此，为了在微生物的功能活性的理解有助于在一个环境，后基因组分析应进行9。因为它揭示了哪些基因被转录，在任何给定的环境Metatranscriptomics，或RNA转录物的分析是有用的。然而，mRNA水平并不总是与它们相应的蛋白质水平由于翻译调控，核糖核酸半衰期，并且可以为每10 mRNA的生成的多个蛋白副本的事实。
由于这些原因metaproteomics现已被公认为对环境微生物学的重要工具。常见metaproteomic分析用散弹枪蛋白质组学的方法，其中的蛋白质在复杂样品的近满装被纯化，并同时进行分析，通常是通过连接zymatic消化成肽和分析质谱仪。随后的串联质谱（MS / MS）“肽指纹识别”用于确定肽序列和原籍潜在蛋白由蛋白质数据库中搜索（综述见11）。蛋白质组的工作已经走过了漫长的道路，在过去25年由于增加的基因组数据的可用性和增加灵敏度和质谱仪，可用于高通量蛋白质鉴定和定量11,12的准确性。因为蛋白质是基因表达的终产物，metaproteomic数据可以帮助确定哪些生物体是活性，在任何给定的环境，什么蛋白他们表达。试图确定一组特定的环境变量会影响生物或社区的表型时，这是有利的。在早期，在海洋中的MS / MS基于metaproteomic研究中使用，以确定在目标特异性蛋白微生物谱系，与第一项研究聚焦在光驱动质子泵遗传工程中SAR11海洋细菌13。最近，比较metaproteomic分析已经阐明复杂的群体之间的差异蛋白质表达谱。例子包括代谢时间的变化在沿海西北大西洋14或南极半岛5的识别 。其他的研究已经在蛋白表达模式描述的变化跨越空间尺度，例如，沿一个地理断面从一个低营养海洋环流到高生产力的沿海上升流系统 15。对于metaproteomics进一步审查建议施奈德等人（2010）9和威廉姆斯等人 （2014）16。目标蛋白质组也已应用在近几年来量化在环境17,18特定的代谢途径的表达。
疗法e为在metaproteomic分析三个主要阶段。第一阶段是样品制备，其包括样品收集，细胞裂解和蛋白的浓度。海洋微生物学样品收集往往造成工作海水的过滤通过一个预过滤器，以去除较大的真核细胞，颗粒和微粒相关的细菌，然后进行过滤的游离活的微生物细胞的捕获，一般与使用0.22μm的盒的过滤单元19,20。这些过滤器incased在一个塑料缸和细胞裂解和蛋白提取协议，可以在过滤器单元内执行将是有价值的工具。一旦获得生物量，将细胞必须被裂解，以允许蛋白质提取。几种方法可以采用，包括盐酸胍裂解21和十二烷基硫酸钠（SDS）基裂解的方法。尽管像SDS洗涤剂非常有效地破坏细胞膜和溶解许多蛋白质种类，concentratiONS低至0.1％，可与下游的蛋白质的消化和质谱分析22干扰。主要关注的是SDS对胰蛋白酶消化效率的负面影响，解决的反相液相色谱法和离子抑制或堆积在离子源23中的电力。
第二阶段是分馏和分析，其中将蛋白进行酶消化，随后通过LC MS / MS分析，结果，可用于确定初始胰蛋白酶肽的一级氨基酸序列上午/ Z裂解谱。各种消化方法可根据所用洗涤剂的类型，以及下游质谱工作流来执行。在我们的协议，1-D PAGE电泳随后从凝胶去除SDS的被利用，以除去任何污染的洗涤剂。蛋白质，是难以溶解，如膜蛋白的分析，需要使用高concen的SDS或其他洗涤剂trations。这导致带SDS-凝胶电泳兼容性问题。如果研究的目标需要的这些坚硬的溶解以溶解的蛋白质，管-凝胶系统可以用来22,24。管 – 凝胶法结合了凝胶基质内的蛋白质，而无需使用电泳的。随后用于溶解任何洗涤剂蛋白质消化之前被除去。
第三个阶段是生物信息学分析。在这个阶段，MS / MS的肽数据被搜索对翻译的核苷酸序列的数据库，以确定哪些肽和蛋白质都存在于样品中。肽的识别取决于其搜索对数据库。海洋metaproteomic数据针对由参考基因组，宏基因组的数据，如全球海洋采样数据集25，以及从未开垦利单细胞扩增的基因组数据库搜索的结果通常neages 26,27。蛋白质鉴定也可以从同一个样品增加通过包含宏基因组序列作为metaproteomic数据推导5。
在这里，我们提供了一个协议，用于通过过滤收集并存储在一个稳定的RNA溶液的肽适用于MS / MS为基础的分析从微生物生物质的产生。这里所描述的协议允许DNA和蛋白质也可以从相同的样品，使所有的步骤导致到蛋白质和DNA沉淀是相同的分离。从实践的角度，需要更少的过滤，因为只有一个过滤器所需的蛋白质和DNA提取。我们还要承认，该协议是通过两个先前公布的协议相结合，改编和修改创建。细胞溶解步骤适于从齐藤等人 （2011）28和在凝胶胰蛋白酶消化成分适于从ShevcheNKO等。 （2007年）29。
Protocol
Representative Results
Discussion
样品保存的关键是metaproteomic研究和以往的研究显示一种RNA稳定的解决方案是之前的蛋白质提取28存储单元一个有用的存储缓冲器。理想情况下，样品将在原地保留处理33,34中否定蛋白表达变化。事实上，原位采样和固定技术已经发展，其允许自主收集和保存的样品的由船部署仪器。但是，获得这些技术并不总是可行的。在于它是不常见的情况下，样品应尽快采集后保?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge Marcos Di Falco for his expertise and advice with the preparation of the samples for nano-LC MS/MS as well as Dr. Zoran Minic from the University of Regina for the LC MS/MS analysis. This work was supported by NSERC (DG402214-2011) and CRC (950-221184) funding. D.C. was supported by Concordia Institute for Water, Energy, and Sustainable Systems and FQRNT.
Materials
| Sterivex -GP 0.22 μm filter unit | Millipore | SVGP01050 | Sampling |
| RNAlater Stabilization Solution | Ambion | AM7021 | Sampling |
| Tris | Bio Basic | 77-86-1 or TB0196-500G | Protein Extraction/ SDS PAGE gel |
| DTT | Sigma-Aldrich | D0632-1G | Protein Extraction |
| SDS | Bio Basic | 15-21-3 | Protein Extraction/ SDS PAGE gel |
| EDTA | Bio Basic | 6381-92-6 | Protein Extraction |
| Glycerol | Fisher Scientific | 56-81-5 | Protein Extraction |
| 10K Amicon Filter | Millipore | UFC801024 | Protein Extraction |
| Methanol | Sigma-Aldrich | 179337-4L | Protein Precipitation |
| Acetone | Fisher Scientific | 67-64-1 | Protein Precipitation |
| MPC Protein Precipitation reagent | Epicenter | mmP03750 | DNA Precipitation |
| 2-Propanol | Fisher Scientific | 67-63-0 | DNA Precipitation |
| Qubit dsDNA BR Assay kit | Life Technologies | Q32850 | DNA Quantification |
| Qubit Protein Assay kit | Life Technologies | Q33211 | Protein Quantification |
| Sucrose | Bio Basic | 57-50-1 | SDS PAGE gel |
| TEMED | Bio Rad | 161-0800 | SDS PAGE gel |
| APS | Bio Rad | 161-0700 | SDS PAGE gel |
| 30% Acrylamide | Bio Rad | 161-0158 | SDS PAGE gel |
| SimplyBlue SafeStain | Invitrogen | LC6060 | SDS PAGE gel |
| Glycine | Bio Rad | 161-0718 | SDS PAGE gel |
| B-mercaptoethanol | Bio Basic | 60-24-2 | SDS PAGE gel |
| Laemmli Sample Buffer | Bio Rad | 161-0737 | SDS PAGE gel |
| Precision Plus Protein Kaleidoscope Ladder | Bio Rad | 161-0375EDU | SDS PAGE gel |
| Acetonitrile | VWR | CABDH6044-4 | In-gel Trypsin digest |
| NH4HCO3 | Bio Basic | 1066-33-7 | In-gel Trypsin digest |
| DTT | Sigma-Aldrich | D0632-1G | In-gel Trypsin digest |
| Formic Acid | Sigma-Aldrich | F0507-500ML | In-gel Trypsin digest |
| HPLC grade H2O | Sigma-Aldrich | 270733-4L | In-gel Trypsin digest |
| Iodoacetamide | Bio Basic | 144-48-9 | In-gel Trypsin digest |
| Trypsin | Promega | V5111 | In-gel Trypsin digest |
| Protein LoBind Tube 1.5ml | Eppendorf | 22431081 | In-gel Trypsin digest |
| 2mL ROBO vial 9mm | Candian Life Science | VT009/C395SB | In-gel Trypsin digest |
| PP BM insert, No spring | Candian Life Science | 4025P-631 | In-gel Trypsin digest |
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