Biology Department, California State University Fresno, 2555 East San Ramon Ave M/S SB73, Fresno, CA 93740, USA.
Int J Mol Sci. 2019 Mar 7;20(5):1172. doi: 10.3390/ijms20051172.
Cell⁻cell communication is vital to multicellular organisms, and distinct types of cellular protrusions play critical roles during development, cell signaling, and the spreading of pathogens and cancer. The differences in the structure and protein composition of these different types of protrusions and their specific functions have not been elucidated due to the lack of a method for their specific isolation and analysis. In this paper, we described, for the first time, a method to specifically isolate distinct protrusion subtypes, based on their morphological structures or fluorescent markers, using laser capture microdissection (LCM). Combined with a unique fixation and protein extraction protocol, we pushed the limits of microproteomics and demonstrate that proteins from LCM-isolated protrusions can successfully and reproducibly be identified by mass spectrometry using ultra-high field Orbitrap technologies. Our method confirmed that different types of protrusions have distinct proteomes and it promises to advance the characterization and the understanding of these unique structures to shed light on their possible role in health and disease.
细胞间通讯对于多细胞生物至关重要，不同类型的细胞突起在发育、细胞信号转导以及病原体和癌症的传播过程中发挥着关键作用。由于缺乏特定的分离和分析方法，这些不同类型的突起的结构和蛋白质组成及其特定功能仍不清楚。本文首次描述了一种使用激光捕获显微切割（LCM）特异性分离基于形态结构或荧光标记的不同突起亚型的方法。结合独特的固定和蛋白质提取方案，我们推动了微蛋白质组学的极限，并证明使用超高场轨道阱技术，通过质谱法可以成功且可重复地鉴定从 LCM 分离的突起中的蛋白质。我们的方法证实了不同类型的突起具有不同的蛋白质组，这有望推进对这些独特结构的表征和理解，以阐明它们在健康和疾病中的可能作用。
