Department of Chemistry & Biochemistry, University of Maryland; Department of Anatomy & Cell Biology, The George Washington University.
Department of Chemistry & Biochemistry, University of Maryland.
J Vis Exp. 2022 Apr 21(182). doi: 10.3791/63586.
Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.
细胞产生组织和器官过程中的分子事件的特征，提高了我们更好地理解正常发育和设计针对疾病的有效疗法的潜力。能够准确识别和定量多种类型和大量蛋白质的技术，将为组织和生物体在时空上发育的分子机制提供仍然缺失的信息。在这里，我们提出了一种基于质谱的方案，该方案能够测量非洲爪蟾（青蛙）胚胎中已鉴定的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已建立的方法之上，这些方法可用于识别、荧光标记、跟踪和采样来自这种脊椎动物发育模型的细胞及其后代（克隆）。使用微采样收集细胞内容物，或者通过解剖或荧光激活细胞分选分离细胞后，提取蛋白质并进行用于下游蛋白质组学分析的处理。使用液相色谱和毛细管电泳为蛋白质检测和高分辨率质谱（HRMS）定量提供可扩展的分离。提供了神经组织定向细胞的蛋白质组学特征的代表性示例。细胞谱系指导的 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，可以窥视脊椎动物发育过程中蛋白质组的时空动态。
