Summary

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

Published: September 15, 2015
doi:

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在凝胶胰蛋白酶消化成分适于从ShevcheNKO等。 (2007年)29。

Protocol

1.准备试剂制备的SDS-萃取溶液:0.1M的Tris-HCl pH为7.5,5%甘油,10mM EDTA的和1%SDS。过滤消毒使用0.22微米的过滤器,并储存在4℃。 备所需的聚丙烯酰胺凝胶的库存试剂。 准备1.5米的Tris-HCl pH值8.8。过滤消毒使用0.22微米的过滤器,并在室温下储存。 的0.5M的Tris-HCl pH 6.8的。过滤消毒使用0.22微米的过滤器,并在室温下储存。 准备10%SDS。过滤消毒使用0.22微米的过滤?…

Representative Results

作为示范,我们进行从表面和叶绿素最大的近海在加拿大北部收集2海水样品的协议。在海上,6-7大号海水通过一个3微米的GF / D预滤器被传递,然后下面的沃尔什等人 20的协议的微生物细胞被收集到0.22微米的筒式过滤器单元。细胞立即储存在RNA稳定溶液直至进一步处理。在返回到实验室,我们执行的协议,因为它是这里提出。将浓缩的细胞裂解物分成;蛋白从90%的体积的沉淀,而DN…

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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Colatriano, D., Walsh, D. A. An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis. J. Vis. Exp. (103), e52827, doi:10.3791/52827 (2015).

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