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Chimica

活蛙 (爪蟾) 胚胎单细胞代谢的微探针毛细管电泳质谱

Published: December 22, 2017
doi:
10.3791/56956
, , ,
1Department of Chemistry,George Washington University, 2Department of Anatomy & Regenerative Biology,George Washington University, 3Department of Chemistry & Biochemistry,University of Maryland, College Park

Summary

我们描述的步骤, 使快速的原位取样的一个小部分, 高精度和最小的入侵使用 capillary-based 微取样, 以促进化学表征的代谢活动的快照活胚胎使用 custom-built 单细胞毛细管电泳和质谱仪平台。

Abstract

单个细胞中小分子的量化为更好地理解胚胎发育的基本过程提供了新的潜能。为了能够直接在活胚胎中进行单细胞调查, 需要新的分析方法, 特别是那些敏感、有选择性、定量、健壮和可伸缩的不同细胞大小。在这里, 我们提出了一个协议, 使原位分析在自由发展的南非爪蛙 (爪蟾) 的单个细胞中的新陈代谢, 这是细胞和发育生物学的一个强有力的模型。这种方法使用毛细管微探针从胚胎中的单个识别细胞中抽出一个定义的部分, 使相邻的细胞保持完好, 以便随后进行分析。通过对高分辨率串联质谱仪进行微尺度毛细管电泳 (CE-ESI) 界面的耦合, 分析采集的细胞含量。这种方法可扩展到不同的细胞大小和兼容的复杂的三维结构发育的胚胎。作为一个例子, 我们证明了微探针单细胞 CE-ESI-ms 能够阐明的新陈代谢细胞异质性, 展开作为祖细胞在胚胎发育过程中产生的后代。除了细胞和发育生物学之外, 这里所描述的单细胞分析协议也可以用于其他细胞大小、细胞类型或动物模型。

Introduction

对胚胎发育的全面了解需要描述在发展中生物体的每个细胞中所展现的所有分子变化。当下一代测序与分子放大, 使深度测量单细胞转录1在开发的系统2,3, 相当少是已知的套件较小的分子在单个胚胎细胞中产生, 包括蛋白质, 特别是代谢物 (分子质量和 #60; ~ 1500 Da)。对内在和外在事件的快速和动态的反应, 代谢充当细胞的分子状态的一个强有力的描述符。因此, 单细胞代谢提高了在早期胚胎中追踪细胞异质性的空间和时间发展的潜能, 并为功能研究寻找新的分子。然而, 如果没有分子放大器可供这些分子, 检测代谢要求特殊灵敏度使用质谱 (MS), 这是技术的选择代谢物分析。

单细胞 MS 是一种技术的集合, 具有足够的灵敏度来测量单个细胞中的代谢物 (见评论4,5,6,7,8,9 ,10,11,12,13,14,15。细胞的重现性取样和代谢物的有效提取对单细胞中代谢物的成功检测至关重要。从爪胚胎中识别出的细胞的全细胞解剖, 使小分子和多肽的表征成为了16。其他方法采用 micropipettes, 以样本的个别活细胞, 其次是检测使用电喷雾电离 (ESI) MS。例如, 代谢物被测量在植物或哺乳动物细胞由单细胞录影 MS17, 压力探针18, 选拔探针19和流体力量显微镜20, 在其他技术之中21, 22、23、24。此外, 化学分离之前, 电离到单细胞 MS 工作流有效地简化了代谢, 从而减轻了潜在的干扰, 在离子生成前检测。重要的是, 分离还提供了复合特定的信息, 以协助分子鉴定。毛细管电泳 (CE) 已被用来检测代谢物在单一解剖25,26或 microsampled27神经元, 捕获小分子表型之间的差异。最近, 我们调整了 CE 到 ESI 串联 MS, 以使从爪蟾16,28的早期胚胎中解剖的单个细胞中的数百种代谢物的微量检测。这些研究揭示了胚胎细胞在发育早期的惊人代谢差异, 并导致发现代谢物与以前未知的发育影响16。

在这里, 我们提供了一个协议, 使检测的代谢物的单细胞直接在活脊椎动物胚胎使用微探针单细胞 CE-ESI-MS29,30。所选择的模型生物体是8至32细胞的x 蟾胚, 虽然这种方法也适用于后期的发展阶段和其他类型的模型生物体。该协议使用高分辨率成像系统指导下的多轴平移控制的锐化毛细管, 以在形态复杂的发育中的胚胎中, 在原位检测到 10 nL 部分的识别细胞。这种微探针可伸缩到较小的细胞, 并在几秒钟内操作, 这是足够快跟踪胚胎细胞谱系。在提取的极性或 apolar 小分子, 如代谢物和多肽, 从收集样本的〜4-5 µL 萃取液, 10 nL 的结果提取是分析在一个 custom-built CE 平台联用的一个 ESI 质谱仪。CE-ESI-MS 平台的构建和运行建立在其他地方所描述的协议之上。31,32轴 CE-ESI 接口是按照其他地方的描述构造的。31此平台在锥喷喷涂系统中保持, 以实现跟踪级别的灵敏度, 并具有在4-5 日志顺序动态范围内进行量化的能力 (相对28、29、30或绝对16)。CE-ESI-MS 平台提供了一个 60 amol 下限检测与8% 相对标准偏差 (RSD) 在定量超过 10 nM 到1µM 为小分子16, 这是足以表征内源代谢物在十。蟾单元格。Microprobed 细胞在胚胎发育的过程中继续分裂30, 允许对细胞代谢进行时空解析分析。事实上, 单细胞 CE-ESI-ms 可以用来发现细胞之间的新陈代谢差异占据了背腹16,29, 动物植物性的16, 和左右28发展轴以及细胞在x 蟾30中, 从一个共同的祖细胞中形成神经组织命中注定的背世系。除了在x 蟾胚30的不同发育阶段对单个胚胎细胞之间的代谢差异进行查询外, 我们预计这里描述的协议适用于广泛的生物分子和单细胞 microsampled 从胚胎发育的不同阶段, 以及其他类型的细胞和模型生物体。此外, 微探针可以用于 microsampling, 而不同的平台与微小的样品相兼容, 可以用于生物分子的分离和/或表征。

Protocol

所有与维护和处理爪蟾有关的协议都是由乔治·华盛顿大学机构动物保育和使用委员会批准的 (IACUC 号。A311)。 1. 取样器具、介质、溶剂和取样盘的制备 准备1x 斯坦伯格的溶液 (SS) 溶解在超纯水 (〜18.2 ω. cm 在25° c) 按以下顺序和在被表明的集中跟随一个标准协议33: 氯化钠 (58.2 毫米), 氯化钾 (0.67 毫米), 硝酸钙 (0.34 毫米), 硫酸镁 (0.83 毫米), 三?…

Representative Results

我们最近使用微探针单细胞 CE-ESI-MS 来表征在自由发展的爪蟾胚29,30中单个识别细胞的代谢物。微探针使快速 (〜5秒/细胞),原位的愿望 10 nL 从一个单独的细胞, 同一细胞的多重愿望, 或几个不同的细胞在相同或后期发展阶段的活胚 (图 1b)。吸气细胞的含量是用合适的溶剂提取的, 然后用 custom-buil…

Discussion

微探针 CE-电喷雾-MS 能够直接表征的代谢物在单细胞的活, 自由发展的胚胎。该方法的核心是两个技术子组件, 即原位毛细管 microsampling 和高敏 CE-电喷雾-MS. 与全细胞解剖相比, 毛细管 microsampling 具有快速操作的优点 (几秒钟与5分钟比较/细胞通过解剖), 与复杂的三维胚胎形态学的相容性, 以及在发育后期形成的更小的细胞的延展性。与解剖不同的是, 微探针取样会使胚胎中的其他细胞保持完好…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家卫生研究院的资助 GM114854 (大法官) 和 CA211635 (大法官), 阿诺德和梅布尔贝克曼基金会贝克曼青年研究员补助金 (大法官), 杜邦青年教授奖 (大法官), 美国大众协会光谱研究奖 (对大法官) 和波斯菊俱乐部基础奖学金 (对 R.M.O. 和 E.P.P.)。本出版物所表达的意见和结论完全是作者的观点, 不一定代表资金来源的官方观点。

Materials

Reagents for Embryo Culture Media
Potasium chloride Fisher Scientific BP 366-1
Magnesium sulfate Fisher Scientific M 65-3
Calcium nitrate Sigma Aldrich C1396
Cysteine MP Biomedicals 101444
Trizma hydrochloride Sigma Aldrich T3253
Trizma base Sigma Aldrich T1503
Sodium chloride Fisher Scientific 5641-212
Name Company Catalog Number Comments
Metabolite Extraction Solvents
Acetonitrile (LC-MS-grade) Fisher Scientific A955
Methanol (LC-MS-grade) Fisher Scientific A456
Water (LC-MS-grade) Fisher Scientific W6
Name Company Catalog Number Comments
Solvents and Standards for CE-ESI-MS
Formic acid (LC-MS-grade) Fisher Scientific A11710X1-AMP
Methanol (LC-MS-grade) Fisher Scientific A456-4
Water (LC-MS-grade) Fisher Scientific W6
Sodium chloride Fisher Scientific 5641-212
Acetylcholine chloride Acros Organics 159170050
Name Company Catalog Number Comments
Microprobe Fabrication Setup
Micropippette puller Sutter Instrument Co. P-1000
Borosilicate capillaries Sutter Instrument Co. B100-50-10
Fine sharp forceps: Dumont #5, Biologie/Dumoxel Fine Science Tools (USA) Inc 11252-30 Corrosion resitant and autoclavable.
Name Company Catalog Number Comments
Microprobe Sampling Setup
Micromanipulator Eppendorf, Hauppauge, NY TransferMan 4r
Stereomicroscope Nikon SMZ18 Should be vibrationally isolated.
Illuminator e.g. Goosenecks Nikon C-FLED2
Microinjector Warner Instrument, Handem, CT PLI-100A
Transfer pipettes (Plastic, disposable) Fisher Scientific 13-711-7M
Petri dish 60 mm and 80 mm Fisher Scientific S08184
Glass Pasteur Pipets ( Borosilicate, disposable) Fisher Scientific 13-678-20A
Centrifuge Thermo Scientific Sorvall Legend X1R
Name Company Catalog Number Comments
CE-ESI-MS Setup
High voltage power supply Spellman CZE1000R The HVPS may be controlled remotely using a low-voltage program generated by a personal computer. Caution: High voltage presents electrical shock hazard; all connective parts must be grounded or carefully shielded to prevent users from accidental exposure.
Syringe pumps (2) Harvard Apparatus 704506
Stereomicroscope Amscope SM-3BZZ Stereomicroscope capable of 4.5× magnification, equipped with an illuminator to monitor the spraying mode of the CE-ESI interface.
XYZ translation stage Thorlabs PT3
XYZ translation stage Custom-built This platform is capable of loading nanoliter-amounts of sample into the separation capillary via hydrodynamic injection and supplying the BGE for CE. Both interfaces described in this work were able to inject 6–10 nL of sample within 1 min into a 1 m separation capillary
Stainless steel sample vials Custom-built
Stainless steel BGE vial Custom-built
Fused silica capillary (40 µm/105 µm ID/OD; 100 cm) Polymicro technologies TSP040105
Fused silica capillary (75 µm/360 µm ID/OD; 100 cm) Polymicro technologies TSP075375
Stainless steel emitter with blunt tips (130/260 µm ID/OD) Hamilton Co. 21031A For better performance, laser-cleave and fine-polish the emitter tip.
Syringes (gas-tight): 500 – 1000 µL Hamilton Co. 1750TTL
Digital multimeter Fluke Fluke 117
High-resolution Mass Spectrometer Bruker Daltonics Maxis Impact HD High-resolution tandem mass spectrometer equipped with an atmospheric-pressure interface configured for ESI
Tunning mixture for mass spectrometer calibration Agilent technologies ESI-L G1969-85000
Data Analysis ver. 4.3 software Bruker Daltonics
Name Company Catalog Number Comments
Ancillary Equipment
Vacuum concentrator capable of operation at 4–10°C Labconco 7310022
Analytical microbalance (XSE105DU) Fisher Scientific 01911005
Freezer (-20 °C) Fisher Scientific 97-926-1
Freezer (-80 °C) Fisher Scientific 88300ASP
Refrigerated Incubator Fisher Scientific 11475126
Vortex-mixer Benchmark BS-VM-1000
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Tags

MicroprobeCapillary ElectrophoresisMass SpectrometrySingle-cell MetabolomicsLive FrogXenopus LaevisEmbryosIn Situ MicrosamplingCell BiologyBiologia dello sviluppoMinimally Invasive SamplingAgarose GelPasteur PipetteBorosilicate CapillariesFlaming/Brown Type Capillary PullerDejellyingSteinberg’s SolutionGonadotropinIn Vitro Fertilization
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Onjiko, R. M., Portero, E. P., Moody, S. A., Nemes, P. Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos. J. Vis. Exp. (130), e56956, doi:10.3791/56956 (2017).
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