| A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry
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Author:
Date:
2020-09-20
[Abstract] Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an ...
[摘要] [抽象]像组蛋白翻译后修饰（PTM）一样，染色质修饰对于调节基因表达和细胞表型的许多其他方面至关重要。液相色谱-质谱联用（LC-MS）已成为最适合大规模分析组蛋白和组蛋白PTM的方法。选定的组蛋白PTM具有已知功能，其异常调节与包括癌症在内的多种疾病有关。但是，组蛋白分析很少用于诊断中，部分是由于通量有限且基于LC-MS的分析的重现性不理想。我们描述了一种使用96孔板进行少于一天的高通量样品制备的工作流程。制备后，使用自动直接进样（DI-MS）方法将样品喷雾到无LC的MS中。每次分析都可以通过45个PTM（甲基化，乙酰化和磷酸化（共151个组蛋白标记）和16个未修饰的组蛋白肽进行组蛋白变体的相对定量。由于没有残留或基于LC的批处理效应，该工作流程允许MS运行少于1分钟，并具有更高的重现性和耐用性。最后，我们描述了一种工程化的肽序列，用于精确监控样品制备的效率，可以在DI-MS运行期间检测到该效率。
[背景] 组蛋白是具有球形头部和N末端尾巴的碱性蛋白质，富含精氨酸和赖氨酸残基。一对典型的组蛋白H2A，H2B，H3和H4（称为核心组蛋白）形成一个八聚体，其周围147 ...
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| Observing Nutrient Gradients, Gene Expression and Growth Variation Using the "Yeast Machine" Microfluidic Device
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Author:
Date:
2020-07-05
[Abstract] The natural environment of microbial cells like bacteria and yeast is often a complex community in which growth and internal organization reflect morphogenetic processes and interactions that are dependent on spatial position and time. While most of research is performed in simple homogeneous environments (e.g., bulk liquid cultures), which cannot capture full spatiotemporal community dynamics, studying biofilms or colonies is complex and usually does not give access to the spatiotemporal dynamics at single cell level. Here, we detail a protocol for generation of a microfluidic device, the “yeast machine”, with arrays of long monolayers of yeast colonies to advance the global understanding of how intercellular metabolic interactions affect the internal structure of colonies ...
[摘要] [摘要 ] 微生物细胞（如细菌和酵母菌）的自然环境通常是一个复杂的社区，在该社区中，生长和内部组织反映了形态发生过程和相互作用，这些过程和相互作用取决于空间位置和时间。虽然大多数研究是在无法捕获完整时空群落动态的简单同质环境（例如，大量液体培养）中进行的，但研究生物膜或菌落却很复杂，通常无法在单个细胞水平上获得时空动态。在这里，我们详细介绍了一种用于生成微流控设备（“酵母机器”）的协议，该协议带有酵母菌落的长单层阵列，以推进对细胞间代谢相互作用如何影响已定义和可定制的空间尺寸内菌落内部结构的全球了解。以酿酒酵母作为模型酵母系统，我们使用“酵母机器”通过追踪荧光标记的己糖转运蛋白来证明葡萄糖梯度的出现。我们进一步量化了菌落内生长速率的表达空间模式和葡萄糖可利用性调控的其他基因的表达。除此之外，我们显示出氨基酸的梯度也在菌落内形成，潜在地打开了类似的方法来研究许多其他营养物和代谢废物的梯度的时空形成。该方法将来可用于在与生态学和进化有关的单细胞分辨率和时标下，破译其他相同物种或更复杂的多物种系统中的远程代谢相互作用，细胞发育和形态发生之间的相互作用。
[背景 ] ...
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