Brioschi Maura, Eligini Sonia, Crisci Mauro, Fiorelli Susanna, Tremoli Elena, Colli Susanna, Banfi Cristina
Centro Cardiologico Monzino, IRCCS, Via Parea 4, 20138, Milan, Italy.
Anal Bioanal Chem. 2014 May;406(12):2817-25. doi: 10.1007/s00216-014-7724-9. Epub 2014 Mar 16.
This paper describes a microproteomic workflow that is useful for simultaneously identifying and quantifying proteins from a minimal number of morphotypically heterogeneous cultured adherent cells. The analytical strategy makes use of laser capture microdissection, an effective means of harvesting pure cell populations, and label-free mass spectrometry. We optimised the workflow with particular reference to cell fixation which is crucial for successful laser-based microdissection and also downstream molecular studies. In addition, we defined the minimum number of cells to be isolated and analysed for satisfactory proteome coverage. To set up this workflow, we choose human monocyte-derived macrophages spontaneously differentiated in vitro. These cells, under our culture conditions, show distinct morphotypes, reminiscent of the heterogeneity observed in tissues in various homeostatic and pathological states, e.g. atherosclerosis. This optimised workflow may provide new insights into biology and pathology of heterogeneous cell in culture, particularly when other cell selection approaches are not suitable.
本文描述了一种微蛋白质组学工作流程，该流程可用于从数量极少的形态学上异质的贴壁培养细胞中同时鉴定和定量蛋白质。该分析策略利用了激光捕获显微切割技术（一种获取纯细胞群体的有效方法）和无标记质谱分析技术。我们特别针对细胞固定优化了工作流程，细胞固定对于基于激光的显微切割以及下游分子研究的成功至关重要。此外，我们确定了为获得满意的蛋白质组覆盖范围而需分离和分析的最少细胞数量。为建立此工作流程，我们选择了在体外自发分化的人单核细胞衍生巨噬细胞。在我们的培养条件下，这些细胞呈现出不同的形态类型，让人联想到在各种稳态和病理状态（如动脉粥样硬化）的组织中观察到的异质性。这种优化的工作流程可能会为培养中的异质细胞的生物学和病理学提供新的见解，尤其是在其他细胞选择方法不适用时。
