JoVE Journal > Chemistry
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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化学

发育（青蛙）胚胎中的细胞谱系引导质谱蛋白质组学

Published: April 21, 2022
doi:
10.3791/63586
, ,
1Department of Chemistry & Biochemistry,University of Maryland, 2Department of Anatomy & Cell Biology,The George Washington University

Summary

在这里，我们描述了脊椎动物非洲 爪蟾 胚胎中具有已知组织命运的细胞谱系的基于质谱的蛋白质组学表征。

Abstract

当细胞产生组织和器官时，分子事件的表征提高了更好地了解正常发育和设计有效的疾病疗法的潜力。能够准确鉴定和定量各种类型和大量蛋白质的技术将提供关于在空间和时间上协调组织和生物体发育的分子机制的信息。在这里，我们提出了一种基于质谱的方案，该协议能够测量非洲 爪 蟾（青蛙）胚胎中已鉴定细胞谱系中的数千种蛋白质。该方法建立在可重复的细胞命运图谱和既定方法的基础上，以从这种脊椎动物发育模型中鉴定，荧光标记，跟踪和采样细胞及其后代（克隆）。使用微量采样或通过解剖或荧光激活细胞分选分离细胞收集细胞内容物后，提取并处理蛋白质以进行自下而上的蛋白质组学分析。液相色谱和毛细管电泳用于通过高分辨率质谱（HRMS）为蛋白质检测和定量提供可扩展的分离。为神经组织命运细胞的蛋白质组学表征提供了代表性的例子。细胞谱系引导的HRMS蛋白质组学适用于不同的组织和生物体。它具有足够的灵敏度、特异性和定量性，可以窥探脊椎动物发育过程中蛋白质组的时空动态。

Introduction

我们对细胞分化以及组织和器官起源的理解是数十年来精心设计的基因及其产物靶向筛选的结果。在重要的细胞事件中，增加我们对所有生物分子及其数量的了解将有助于解开控制脊椎动物身体计划的空间和时间模式的分子机制。能够进行分子扩增和测序的技术现在能够定期报告大量基因和转录本，支持基础生物学和转化研究中的假设驱动研究。为了理解发展中的系统，转录和翻译之间的复杂关系主张直接分析多种蛋白质及其翻译后修饰。使用 体外 生物系统（如诱导多能干细胞）的全球蛋白质组学开始描绘组织诱导的机制1，2。在复杂的生物中，例如脊椎动物胚胎，发育依赖于空间和时间背景下的形态原梯度3。因此，随着细胞分化形成专门的组织（如神经组织），获得蛋白质组学变化的知识，为解锁控制正常和缺陷发育的分子程序和指导下一代疗法提供了一把钥匙。

脊椎动物南非爪蛙（非洲爪蟾）是细胞和发育、神经和再生生物学的成熟模型。约翰·戈登爵士（Sir John Gurdon）因发现体细胞核的多能性而获得2012年诺贝尔生理学或医学奖4，5，强调了该模型对基础和转化研究发现的重要性。非洲爪蟾胚胎在母体外部发育，从而促进细胞、细胞克隆和基因表达在不同发育阶段的直接操作。不对称的色素沉着和刻板的细胞分裂使得能够绘制出来自 16-6 和 32 细胞7，8 期胚胎的可重复命运图。对于基于高分辨率质谱（HRMS）的蛋白质组学，该模型的其他优点包括相对较大的尺寸（直径~1毫米），这会产生丰富的蛋白质含量进行分析（早期切割阶段胚胎~130μg，16细胞胚胎的单细胞中~10μg蛋白质含量）9，10。

目前，HRMS是检测蛋白质的首选领先技术。该技术能够直接、灵敏、特异性地检测和定量多种（通常是数百到数千种不同的蛋白质）11。HRMS自下而上的蛋白质组学涉及一系列相互关联的步骤。从细胞/组织样品中提取后，用蛋白水解酶（如胰蛋白酶（自下而上的蛋白质组学））消化蛋白质。所得肽根据其不同的物理化学性质进行分离，包括疏水性（反相液相色谱，LC），净电荷（离子交换色谱），大小（尺寸排阻色谱）或电泳迁移率（毛细管电泳，CE）。然后对肽进行充电（电离），通常使用电喷雾电离（ESI），并通过串联HRMS通过气相碎裂检测和测序肽离子。所得肽数据映射到所研究生物体的蛋白质组。由于蛋白质特异性（蛋白型）肽离子信号强度与浓度相关，蛋白质定量可以无标记或基于标记（多重定量）进行。HRMS蛋白质组学产生了有关所研究系统分子状态的丰富信息资源，允许生成假设和后续功能研究。

Figure 1
图 1：时空可扩展的蛋白质组学，使细胞谱系引导发育（青蛙）胚胎中的 HRMS 蛋白质组学成为可能。 （A）在平移阶段（4）的控制下，使用体视显微镜（2）注射已识别的细胞（插图）的样品（1）用于注射已识别的细胞（插图）。（B）对16细胞胚胎中鉴定的左D11细胞进行亚细胞采样。（C）从16细胞胚胎中解剖整个D11细胞。（D）从32细胞胚胎中对左右D111后代进行荧光（绿色）示踪，以指导解剖原肠胚中的神经外胚层（NE）（第10阶段），并使用FACS从蝌蚪中分离后代组织。比例尺：胚胎200μm，小瓶1.25mm。数字经参考文献15，19，21，59许可改编。请点击此处查看此图的大图。

这里介绍的方案能够基于HRMS对发育中的X. laevis胚胎中鉴定的细胞/组织中的大量蛋白质进行定量。该方法建立在准确的细胞鉴定、可重复的细胞命运图谱和在该生物模型中跟踪细胞谱系的既定方法之上6，7，8。如图1所示，我们通过采用全细胞解剖或毛细管微量采样来抽吸细胞内容物来研究来自单细胞的蛋白质组。监测细胞的谱系使我们能够研究蛋白质组的时空演变，因为细胞在原肠胚形成过程中形成组织。通过注射与惰性葡聚糖或mRNA偶联的荧光团来标记细胞后代，以用于荧光蛋白（例如，绿色荧光蛋白或GFP）。标记的后代在所需的发育时间点分离。在原肠胚形成过程中，可以通过解剖分离紧密聚集的细胞克隆。原肠胚形成后，由于迁移运动，细胞克隆可能分布在胚胎内，并且可以通过荧光激活细胞分选（FACS）从解离组织中分离出来。这些细胞和组织中的蛋白质通过自下而上的蛋白质组学进行测量，采用HPLC或CE进行分离，ESI串联HRMS进行鉴定。细胞谱系引导的HRMS蛋白质组学可扩展到胚胎内的不同细胞大小和谱系，并且具有特异性、灵敏度和定量性。通过此处显示的精选示例，我们还证明了该协议具有可扩展性，并且广泛适用于不同类型的细胞和细胞谱系。

Figure 2
图 2：生物分析工作流程。 显微解剖和毛细管抽吸，或FACS有助于细胞和克隆蛋白含量的采样。使用电喷雾电离 （ESI） 高分辨率质谱 （HRMS） 去除丰度蛋黄蛋白并通过毛细管电泳 （CE） 或纳流液相色谱 （LC） 增强鉴定 （ID） 灵敏度进行分离。量化揭示了失调，结合基因本体（GO）提供的信息，为假设驱动的研究提供了新的信息。数字经参考文献15许可改编。 请点击此处查看此图的大图。

Protocol

确保非洲 爪蟾 成年蛙的人道维护和处理的所有协议均已获得马里兰大学帕克分校机构动物护理和使用委员会的批准（批准号R-DEC-17-57和R-FEB-21-07）。 1. 准备解决方案 用于胚胎学准备1x，0.5x和0.2x斯坦伯格溶液（SS），然后按照标准方案12将它们高压灭菌（120°C20分钟）至无菌。 按照标准方案在灭菌的 1x SS 中制备 3% （w/…

Representative Results

该协议能够研究单细胞中的蛋白质及其谱系，因为它们在X. laevis胚胎中建立组织。图1说明了该方法在神经组织命运细胞和胚胎中新诱导的神经外胚层中研究蛋白质的一种应用。如图1A所示，生物分析工作流程集成了传统的细胞和发育生物学工具，用于鉴定、注射/吸出细胞和收集标本。图1B显示了使用显微注射器对体内</e…

Discussion

该协议能够表征 非洲爪蟾 物种胚胎中已鉴定细胞谱系中的蛋白质表达。该方法源于HRMS，结合了分子鉴定的精湛特异性，无需分子探针即可进行多蛋白检测的能力（通常是数百到数千种不同的蛋白质）以及定量能力。对细胞和发育（神经）生物学中经典工具和工作流程的适应性将HRMS蛋白质组学扩展到令人兴奋的应用，包括脊椎动物X . laevis 胚胎中干细胞分化的整体表征。

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Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢Jie Li（马里兰大学帕克分校）关于胚胎解离和FACS的宝贵讨论。我们感谢Vi M. Quach和Camille Lombard-Banek在先前的研究中为样品制备和数据收集提供的帮助，这些研究举例说明了本协议中强调的蛋白质组学应用。这项工作的部分内容得到了美国国家科学基金会的支持，奖励号为IOS-1832968 CAREER（授予P.N.），美国国立卫生研究院的奖励号为R35GM124755（授予P.N.），马里兰大学 – 国家癌症研究所合作计划（授予P.N.）和COSMOS俱乐部基金会研究奖（授予ABB和L.R.P.）。

Materials

Acetonitrile (LC-MS-grade) Fisher Scientific A955
Agarose ThermoFisher Scientific R0492
Ammonium bicarbonate Fisher Scientific A643-500
Analytical Column Thermo Scientific 164941
Analytical microbalance Mettler-Toledo XSE105DU
Automatic peptide fractionation platform Agilent 1260 Infinity II
Borosilicate Capillaries Sutter Instruments Co. B100-50-10
Borosilicate Capillaries (for making Emmitters) Sutter Instruments B100-75-10
C18 spin columns (for desalting) ThermoFisher Scientific 89870
Camera ro monitor electrospray Edmund Optics Inc. EO-2018C
Combretastatin A4 Millipore Sigma C7744
Commercial CESI system AB SCIEX CESI
(Cyclohexylamino)-1-propanesulfonic acid (CAPS) VWR 97061-492
Cytochalasin D Millipore Sigma C8273
Dextran, Alexa Fluor 488; 10,000 MW, Anionic, Fixable ThermoFisher Scientific D22910
Diothiothreitol Fisher Scientific FERR0861
Dumont #5 Forceps Fine Science Tools 11252-30
EDTA Fisher Scientific AAJ62786AP
Epifluorescence light source Lumencore AURA III
Eppendorf LoBing microcentrifuge  tubes: protein Fisher Scientific 13-698-793
Formic acid (LC-MS-grade) Fisher Scientific A117-50
Freezer (-20 °C) Fisher Scientific 97-926-1 
Freezer (-80 °C) Thermo Scientific TSX40086A
Fused silica capillary Molex 1088150596
Heat Block Benchmark BSH300
High pressure liquid Chromatography System ThermoFisher Scientific Dionex Ultimate 3000 RSLC nanosystem
High voltage power supply Spellman CZE1000R
High-resolution Mass Spectrometer ThermoFisher Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer
HPLC caps Thermo Scientific C4013-40A
HPLC Vials Thermo Scientific C4013-11
Illuminator e.g. Goosenecks Nikon C-FLED2
Ingenuity Pathway Analysis Qiagen
Iodoacetamide Fisher Scientific AC122275000
Methanol (LC-MS-grade) Fisher Scientific A456
Methanol (LC-MS-grade) Fisher Scientific A456-4
Microcapillary puller Suttor Instruments P-2000
Microinjector Warner Instrument, Handem, CT PLI-100A
Micropippette puller Sutter Instruments Co. P-1000
MS data analysis software, commercial ProteomeDiscoverer
MS data analysis software, opensource MaxQuant
non-idet 40 substitute Millipore Sigma 11754599001
Petri dish 60 mm and 80 mm Fisher Scientific S08184
Pierce 10 µL bed Zip-tips (for desalting) ThermoFisher Scientific 87782
Pierce bicinchoninic acid protein assay kit ThermoFisher Scientific 23225
Pierce quantitative colorimetric peptide assay ThermoFisher Scientific 23275
Pierce Trypsin Protease (MS Grade) Fisher Scientific PI90058
Protein LoBind vials Eppendorf 0030108434
, 0030108442
Refrigerated Centrifuge Eppendorf 5430R
Refrigerated Incubator Thermo Scientific PR505755R/3721
sodium isethionate Millipore Sigma 220078
sodium pyrophosphate Sigma Aldrich 221368-100G
Stainless steel BGE vial Custom-Built
Stainless steel sample vials Custom-Built
Stereomicroscope (objective 10x) Nikon SMZ 1270, SZX18
Sucrose VWR 97063-790
Syringe pumps (2) Harvard Apparatus 704506
Syringes (gas-tight): 500–1000 µL Hamilton 1750TTL
Transfer pipettes (Plastic, disposable) Fisher Scientific 13-711-7M
Trap Column Thermo Scientific 164750
Tris-HCl (1 M solution) Fisher Scientific AAJ22638AP
Vacuum concentrator capable of operation at 4–10 °C Labconco 7310022
Vortex-mixer Benchmark BS-VM-1000
Water (LC-MS-grade) Fisher Scientific W6
Water (LC-MS-grade) Fisher Scientific W6
XYZ translation stage Thorlabs PT3
XYZ translation stage Custom-Built
DOWNLOAD MATERIALS LIST

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Baxi, A. B., Pade, L. R., Nemes, P. Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo. J. Vis. Exp. (182), e63586, doi:10.3791/63586 (2022).
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