Utilization of Laser Capture Microdissection Coupled to Mass Spectrometry to Uncover the Proteome of Cellular Protrusions.
Utilization of Laser Capture Microdissection Coupled to Mass Spectrometry to Uncover the Proteome of Cellular Protrusions.
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DOI：
10.1007/978-1-0716-1178-4_3
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Gousset K
中科院分区：
文献类型：
--
作者：
Gordon A;Gousset K
激光捕获显微切割(LCM)提供了一种快速、特异和通用的方法来分离和丰富混合种群和/或亚细胞结构中的细胞，用于进一步的蛋白质组学研究。此外，质谱学(MS)可以快速、准确地从少量样本中生成差异蛋白质表达谱。尽管细胞突起--如隧道纳米管、丝状足突、生长锥体、侵袭突起等--参与了吞噬或癌细胞侵袭等基本的生理和病理活动，但由于其脆弱性和暂时性，对其蛋白质组成的研究进展缓慢。这里描述的方法，结合LCM和MS，已经被设计来识别不同细胞突起的蛋白质组。首先，用一种新的固定方法固定细胞，以保存细胞突起，这些突起通过LCM分离。其次，对浓缩样品中蛋白质的提取进行了优化，使固定剂脱交，从而提高了蛋白质的MS鉴定效率。这种方法高效的蛋白质回收和高质量的样品质量使这些小而多样的亚细胞结构的蛋白质图谱成为可能。
Laser Capture Microdissection (LCM) provides a fast, specific, and versatile method to isolate and enrich cells in mixed populations and/or subcellular structures, for further proteomic study. Furthermore, Mass Spectrometry (MS) can quickly and accurately generate differential protein expression profiles from small amounts of samples. Although cellular protrusions—such as tunneling nanotubes, filopodia, growth cones, invadopodia, etc.—are involved in essential physiological and pathological actions such as phagocytosis or cancer-cell invasion, the study of their protein composition is progressing slowly due to their fragility and transient nature. The method described herein, combining LCM and MS, has been designed to identify the proteome of different cellular protrusions. First, cells are fixed with a novel fixative method to preserve the cellular protrusions, which are isolated by LCM. Next, the extraction of proteins from the enriched sample is optimized to de-crosslink the fixative agent to improve the identification of proteins by MS. The efficient protein recovery and high sample quality of this method enables the protein profiling of these small and diverse subcellular structures.