土壤样品多涨价可能分析的 MPLEx 协议
Summary
提出了一种从单个土壤样品中同时提取代谢物、蛋白质和脂质的协议, 允许缩短样品准备时间, 并对数量有限的样品进行多涨价可能质谱分析。
Abstract
以质谱 (MS) 为基础的综合 metaproteomic、metabolomic 和 lipidomic (多涨价可能) 研究正在改变我们对环境和生物系统中微生物群落的理解和表征能力。这些测量甚至能够增强对复杂土壤微生物群落的分析, 这是迄今为止已知的最复杂的微生物系统。然而, 多涨价可能分析确实有样本准备挑战, 因为每个涨价可能研究通常需要分离提取, 从而大大放大所需的准备时间和样本量。为了解决这一局限性, 通过调整溶剂基方法, 建立了从同一土壤样品中同时提取代谢物、蛋白质和脂质 (MPLEx) 的 3-1 方法。这种 MPLEx 协议已被证明是简单和健壮的许多样本类型, 即使是用于有限数量的复杂土壤样品。MPLEx 方法还大大启用了快速的多涨价可能测量, 以更好地了解每个微生物群落的成员, 同时评估发生在生物和环境扰动上的变化。
Introduction
评价土壤微生物群落对了解碳循环和气候变化具有重要意义。然而, 最近的研究突出了一些困难, 例如在不同土壤类型的微生物群中缺乏有序的基因组, 以及检测到的许多蛋白质的未知功能。这些挑战的结果是由于土壤是迄今为止已知的最复杂的微生物群落1,2,3。多涨价可能分析结合了宏基因组、metatranscriptomic、metaproteomic、metabolomic 和 lipidomic 研究的结果, 最近在许多土壤研究中得到了实施, 以更好地了解目前的微生物, 而获取有关由于环境扰动而发生的分子变化的全面信息1,4,5。多涨价可能研究的一个挑战是, 以质谱 (MS) 为基础的 metaproteomic、metabolomic 和 lipidomic 测量通常要求每个涨价可能的特定提取过程为 MS兼容6, 7,8,9. 当只有有限数量的样本可用时, 这些精确的程序使得它们的实施极其困难或不可能实现。这些挑战促使我们研究一种同时代谢物、蛋白质和脂质提取 (MPLEx) 方法, 能够使用体积较小的样本量或质量, 提高准确度, 并为所有三种分析提供更快的样品准备。10. 迄今为止, 没有任何替代土壤提取程序可以实现所有这些目标。
为了实现对单个土壤样品的全局多涨价可能分析, 采用了基于氯仿、甲醇和水分离的有机溶剂萃取协议 10.此方法最初是为总脂提取而开发的9,11和最近被修正了为同时提取代谢物, 蛋白质和油脂从一个样品12,13 ,14,15,16,17,18,19,20,21,22, 23,24,25,26,27,28,29,30, 启用较少的样本数量和实验性变异性10。在 MPLEx 协议中, 氯仿不与水混溶, 这为将样品组分三相化学分离为不同组分提供了依据。因此, 顶水相包含亲水性代谢物, 其次是蛋白质盘, 然后是底部氯仿阶段的脂质层 (图 1)。当 MPLEx 适用于大多数土壤时, 微粒碎片堆积在取样管的底部, 并且在收集所有层后可以被丢弃。然而, 每个土壤类型可以不同, 在高度有机土壤例如泥煤, 土壤残骸停留在中间层数并且不下落到取样管的底部。MPLEx 提供了几个优势, 当隔离多个分子类型从相同的样本, 如 1) 较小的样本量可用于多涨价可能分析, 2) 多涨价可能提取从相同的样本减少总体实验变异性, 并3) 可以更快地为更高吞吐量研究10编写更多的样本。这些好处对提供更好的测量能力以评估土壤样品及其复杂的微生物群落是至关重要的。
Protocol
注: 非常潮湿的土壤可以在萃取前冻干, 而不损害萃取效果。湿土壤也可以使用, 但应考虑当添加试剂在特定比率。 注: 建议使用20克的干土重量每萃取, 这必须在两个50毫升管 (最大10克土壤每50毫升管)。抽取可以根据可用样品向上或向下缩放。 注: 干燥土壤样品可以通过3毫米筛筛, 以融汇和去除小的根和岩石。不要筛过潮湿的土壤样品, 因为样品会被卡在?…
Representative Results
当 MPLEx 协议被用来从堪萨斯当地草原土壤中提取分子 (软土土壤) 时, 三项分析提供了3376多肽、105脂和102极性代谢物 (所有独特的证明) 的结果。虽然 MPLEx 协议已建立为一般提取脂质和代谢物12,13,14,15,16,17,<sup class="xre…
Discussion
重要的是要注意的是, 并非所有的实验室都有相同的可用设备, 所以某些方法, 例如裂解步骤, 可以适应。在这里, 我们使用涡流和 sonicating, 但使用一个大型的50毫升珠搅拌器将工作。如果冻干机的集热器温度可为-105 摄氏度, 则可以在氮气流下干燥样品。土壤类型也有很大变化, 可以包括沙子、淤泥、粘土、泥炭和沃土 (等等), 它们也可以根据 pH 值、盐度和有机质的丰富程度而变化。这些差异…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢内森. 约翰逊在准备这些数字方面的协助。这项研究得到了由美国能源部生物和环境研究局 (基因组科学计划) 资助的泛组学计划的支持, 微生物 (薄荷) 实验室指导研究开发倡议在太平洋西北国家实验室, 以及国家卫生研究院国家环境卫生科学研究所 (R01 ES022190) 和 NIH (P42 ES027704)。KEBJ 感谢 R21 HD084788 为开发和验证新的多涨价可能提取技术提供财政支持。这项工作是在格雷斯威利环境分子科学实验室 (EMSL), 在太平洋西北国家实验室 (PNNL) 的能源部国家科学用户设施进行的。PNNL 是由巴特利在合同 DE-AC06-76RL01830 下为能源部管理的多方案国家实验室。
Materials
| Chloroform | Sigma-Aldrich | 650498 | Stored at -20°C !Caution chloroform has acute potential health effects, skin irritation and possible chemical burns, irritation to the respiratory system, may affect the kidneys, liver, heart. Wear suitable protective glasses, clothing and gloves, work in a fume hood. |
| Methanol | Sigma-Aldrich | 34860 | Stored at -20°C !Caution Methanol may cause respiratory tract, skin and eye irritation, may damage the nerves, kidneys and liver. Wear suitable protective glasses, clothing and gloves, work in a fume hood. |
| Purified water from Millipore | Milli-Q | Water purification system. | |
| Sodium dodecyl sulfate | Sigma-Aldrich | L6026 | !Caution SDS causes acute toxicity and is flammable. It is a skin, eye and airway irritant. Wear gloves and safety glasses. |
| Soil protein extraction kit | MoBio, NoviPure Soil Protein Extraction Kit, Qiagen | 30000-20 | |
| DL-dithiothreitol | Sigma-Aldrich | 43815 | |
| 1M Trizma HCL | Sigma-Aldrich | T2694 | |
| Trichloroacetic acid | Sigma-Aldrich | T0699 | !Caution TCA is caustic, toxic and may cause skin burns. Wear gloves and safety glasses. |
| Acetone | Sigma-Aldrich | 650501 | Stored at -20°C !Caution Acetone may cause respiratory tract and skin and eye irritation. Flammable liquid and vapor. Wear safety glasses gloves and a lab coat, work in a fume hood. |
| Urea | Sigma-Aldrich | 208884 | !Caution Urea is an eye and skin irritant, use gloves and safety glasses |
| Ammonium bicarbonate | Fluka | 09830 | |
| Trypsin | Promega | V528A | 20µg vials |
| Bicinchoninic acid protein assay kit | Pierce | 23227 | |
| Ammonium Formate | Sigma-Aldrich | 09735 | |
| Acetonitrile | Sigma-Aldrich | 34998 | !Caution Acetonitrile is a skin and eye irritant. Highly flammable. Wear gloves and safety glasses. Work in a fume hood. |
| Trifluoroacetic acid | Sigma-Aldrich | T6508 | !Caution TFA is extremely hazardous in case of skin contact, eye contact, ingestion and inhalation. May produce tissue damage particularly on mucous membranes of eyes, mouth and respiratory tract. Skin contact may produce burns. Wear gloves, lab coat, safety glasses and work in a fume hood. |
| Methoxyamine hydrochloride | Sigma-Aldrich | 226904 | !Caution Methoxyamine hydrochloride causes severe burns and serious damage to eyes, may cause sensitization by skin contact. Wear safety glasses, gloves and lab coat, work in a fume hood. |
| Pyridine | Sigma-Aldrich | 270970 | !Caution Pyridine can cause skin and eye irritation, central nervous system depression. Vapor may cause flash fire. Wear safety glasses, gloves and lab coat, work in a fume hood. |
| N-Methyl-N-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane | Sigma-Aldrich | 69478 | !Caution MSTFA + 1% TMCS can cause skin corrosion, serious eye damage and specific target organ toxicity. Flammable liquid and vapor. Wear safety glasses, gloves and lab coat, work in a fume hood. |
| Potassium chloride | Sigma-Aldrich | P9541 | |
| Milli-Q water purification system | Millipore | model MPGP04001 | |
| Vortex | Scientific Industries | SI-0236 | Vortex Genie 2 |
| Probe sonicator | FisherBrand | model FB505 | |
| Refrigerated centrifuge | Eppendorf | model 5810R | |
| 50mL tube swinging bucket rotor | Eppendorf | A-4-44 | |
| 50mL fixed angle rotor | Eppendorf | FA-45-6-30 | |
| Balance | OHAUS | model V22PWE150IT | |
| Serological pipette controller | Eppendorf | 12-654-100 | |
| 10mL, 25mL glass serological pipettes | FisherBrand | 13-678-27F, 13-678-36D | |
| Thermomixer with Thermotop | Eppendorf | 5382000015, 5308000003 | |
| 0.9 – 2.0 mm blend stainless steel beads | NextAdvance | SSB14B | |
| 0.15 mm garnet beads | MoBio | 13122-500 | |
| Magnetic stir plate | FisherBrand | 11-100-16SH | |
| Magnetic stir bar | FisherBrand | 14512130 | |
| pH paper strips, pH range 0–14 | FisherBrand | M95903 | |
| 15mL, 50mL conical polypropylene centrifuge tube | Genesee Scientific | 21-103 21-108 | chloroform compatible |
| 50mL vortex attachment | MoBio | 13000-V1-50 | |
| Ice bucket | FisherBrand | 02-591-44 | |
| 27.25x70mm glass vials | FisherBrand | 03-339-22K | |
| Breathe Easier plate membranes | Midwest Scientific | BERM-2000 | |
| Alcohol wipes | Diversified Biotech | BPWP-1000 | |
| Heater shaker incubator | Benchmark, Incu-Shaker Mini | ||
| Analog rotisserie tube rotator | SoCal BioMed, LLC | 82422001 | |
| Filter-Aided-Sample-Prep kit | FASP; Expedeon | 44250 | |
| Microplate reader | Biotek, EPOCH | ||
| -20 Degree Celsius Freezer | Fisher | 13986149 | |
| -80 Degree Celsius Freezer | Stirling Ultracold | SU78OUE | |
| Q-Exactive ion trap mass spectrometer | Thermo Scientific | ||
| Agilent 7890A gas chromatograph coupled with a single quadrupole 5975C mass spectrometer | Agilent Technologies, Inc. | ||
| LTQ-Orbitrap Velo | Thermo Scientific | ||
| Waters NanoEquityTM UPLC system | Millford, MA | ||
| 250mL media bottle | FisherBrand | 1395-250 | |
| Waters vial | Waters | 186002805 | |
| Glass MS sample vial and inserts | MicroSolv | 9502S-WCV, 9502S-02ND | |
| Glass HPLC vial and snap caps | MicroSolv | 9512C-0DCV, 9502C-10C-B | |
| HPLC 96-well plate | Agilent | 5042-6454 | |
| Large glass vial 27.25x70mm | FisherBrand | 03-339-22K | |
| Lyophilizer | Labconco | 7934021 | |
| Polished stainless steel flat head spatula | Spoonula; FisherBrand | 14-375-10 | |
| Kim wipes | Kimberly-Clark | 34721 | |
| XBridge C18, 250×4.6 mm, 5 μM with 4.6×20 mm guard column | Waters | 186003117, 186003064 | |
| Agilent 1100 series HPLC system | Agilent Technologies | G1380-90000 | |
| 1.7mL centrifuge tube | Sorenson | 11700 | |
| Hamilton Glass Syringes, 5mL, 50µL and 250µL | Hamilton | 81517, 80975, 81175 | |
| Pasteur Pipettes | FisherBrand | 13-678-20A | |
| Pasteur Pipette Bulbs | Sigma-Aldrich | Z111597 | |
| Bath Sonicator | Branson 1800 Ultrasonic Cleaner | ||
| Vacuum Centrifuge | Labconco Centrivap Acid-Resistant Concentrator System | ||
| MicroSpin Columns, C18 Silica | The Nest Group | SEM SS18V | |
Riferimenti
- Hultman, J., et al. Multi-omics of permafrost, active layer and thermokarst bog soil microbiomes. Nature. 521, 208-212 (2015).
- White, R. A., et al. Moleculo long-read sequencing facilitates assembly and genomic binning from complex soil metagenomes. mSystems. 1, (2016).
- White, R. A., Callister, S. J., Moore, R. J., Baker, E. S., Jansson, J. K. The past, present and future of microbiome analyses. Nat Protoc. 11, 4-8 (2016).
- Ritchie, M. D., Holzinger, E. R., Li, R., Pendergrass, S. A., Kim, D. Methods of integrating data to uncover genotype-phenotype interactions. Nat Rev Genet. 16, 85-97 (2015).
- Jansson, J. K., Baker, E. S. A multi-omic future for microbiome studies. Nat Microbiol. 1, (2016).
- Domon, B., Aebersold, R. Options and considerations when selecting a quantitative proteomics strategy. Nat Biotechnol. 28, 710-721 (2010).
- Marx, V. Targeted proteomics. Nat Methods. 10, 19-22 (2013).
- Roberts, L. D., Souza, A. L., Gerszten, R. E., Clish, C. B. Targeted metabolomics. Curr Protoc Mol Biol. , (2012).
- Folch, J., Lees, M., Sloane Stanley, G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 226, 497-509 (1957).
- Nakayasu, E. S., et al. MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses. mSystems. 1, (2016).
- Bligh, E. G., Dyer, W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 37, 911-917 (1959).
- Pomraning, K. R., et al. Multi-omics analysis reveals regulators of the response to nitrogen limitation in Yarrowia lipolytica. BMC Genomics. 17, 138 (2016).
- Tisoncik-Go, J., et al. Integrated Omics Analysis of Pathogenic Host Responses during Pandemic H1N1 Influenza Virus Infection: The Crucial Role of Lipid Metabolism. Cell Host Microbe. 19, 254-266 (2016).
- Kyle, J. E., et al. Uncovering biologically significant lipid isomers with liquid chromatography, ion mobility spectrometry and mass spectrometry. Analyst. 141, 1649-1659 (2016).
- Lovelace, E. S., et al. Silymarin Suppresses Cellular inflammation by inducing reparative stress signaling. J Nat Prod. 78, 1990-2000 (1990).
- Kim, Y. M., et al. Diel metabolomics analysis of a hot spring chlorophototrophic microbial mat leads to new hypotheses of community member metabolisms. Front Microbiol. 6, 209 (2015).
- Pomraning, K. R., et al. Comprehensive Metabolomic, Lipidomic and microscopic profiling of Yarrowia lipolytica during lipid accumulation identifies targets for increased lipogenesis. PLoS One. 10, e0123188 (2015).
- Huang, E. L., et al. The fungus gardens of leaf-cutter ants undergo a distinct physiological transition during biomass degradation. Environ Microbiol Rep. 6, 389-395 (2014).
- Deatherage Kaiser, B. L., et al. A Multi-Omic View of Host-Pathogen-Commensal Interplay in Salmonella-Mediated Intestinal Infection. PLoS One. 8, e67155 (2013).
- Kim, Y. M., et al. Salmonella modulates metabolism during growth under conditions that induce expression of virulence genes. Mol Biosyst. 9, 1522-1534 (2013).
- Ansong, C., et al. A multi-omic systems approach to elucidating Yersinia virulence mechanisms. Mol Biosyst. 9, 44-54 (2013).
- Bordbar, A., et al. Model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation. Mol Syst Biol. 8, 558 (2012).
- Hu, Z. P., et al. Metabolomic response of human skin tissue to low dose ionizing radiation. Mol Biosyst. 8, 1979-1986 (2012).
- Perera, R., et al. Dengue virus infection perturbs lipid homeostasis in infected mosquito cells. PLoS Pathog. 8, e1002584 (2012).
- Gao, X., et al. A reversed-phase capillary ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method for comprehensive top-down/bottom-up lipid profiling. Anal Bioanal Chem. 402, 2923-2933 (2012).
- Sorensen, C. M., et al. Perturbations in the lipid profile of individuals with newly diagnosed type 1 diabetes mellitus: Lipidomics analysis of a Diabetes Antibody Standardization Program sample subset. Clin Biochem. 43, 948-956 (2010).
- Diamond, D. L., et al. Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics. PLoS Pathog. 6, e1000719 (2010).
- Alquier, T., et al. Deletion of GPR40 impairs glucose-induced insulin secretion in vivo in mice without affecting intracellular fuel metabolism in islets. Diabetes. 58, 2607-2615 (2009).
- Ding, J., et al. Application of the accurate mass and time tag approach in studies of the human blood lipidome. J Chromatogr B Analyt Technol Biomed Life Sci. 871, 243-252 (2008).
- Rasmussen, A. L., et al. Systems virology identifies a mitochondrial fatty acid oxidation enzyme, dodecenoyl coenzyme A delta isomerase, required for hepatitis C virus replication and likely pathogenesis. J Virol. 85, 11646-11654 (2011).
- Manza, L. L., Stamer, S. L., Ham, A. J., Codreanu, S. G., Liebler, D. C. Sample preparation and digestion for proteomic analyses using spin filters. Proteomics. 5, 1742-1745 (2005).
- Wisniewski, J. R., Zougman, A., Nagaraj, N., Mann, M. Universal sample preparation method for proteome analysis. Nat Methods. 6, 359-362 (2009).
- Zhou, J. Y., et al. Simple sodium dodecyl sulfate-assisted sample preparation method for LC-MS-based proteomics applications. Anal Chem. 84, 2862-2867 (2012).
- Anderson, J. C., et al. Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae. Proc Natl Acad Sci U S A. 111, 6846-6851 (2014).
- Chourey, K., et al. Direct cellular lysis/protein extraction protocol for soil metaproteomics. J Proteome Res. 9, 6615-6622 (2010).
- Kim, S., Gupta, N., Pevzner, P. A. Spectral probabilities and generating functions of tandem mass spectra: a strike against decoy databases. J Proteome Res. 7, 3354-3363 (2008).
- Kim, S., Pevzner, P. A. MS-GF+ makes progress towards a universal database search tool for proteomics. Nat Commun. 5, 5277 (2014).
- Cole, J. K., et al. Phototrophic biofilm assembly in microbial-mat-derived unicyanobacterial consortia: Model systems for the study of autotroph-heterotroph interactions. Front Microbiol. 5, (2014).
- Isaacson, T., et al. Sample extraction techniques for enhanced proteomic analysis of plant tissues. Nat Protoc. 1, 769-774 (2006).
Citazione di questo articolo
Nicora, C. D., Burnum-Johnson, K. E., Nakayasu, E. S., Casey, C. P., White III, R. A., Roy Chowdhury, T., Kyle, J. E., Kim, Y., Smith, R. D., Metz, T. O., Jansson, J. K., Baker, E. S. The MPLEx Protocol for Multi-omic Analyses of Soil Samples. J. Vis. Exp. (135), e57343, doi:10.3791/57343 (2018).