A mass spectrometry-based workflow for the proteomic analysis of in vitro cultured cell subsets isolated by means of laser capture microdissection
A mass spectrometry-based workflow for the proteomic analysis of in vitro cultured cell subsets isolated by means of laser capture microdissection
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DOI：
10.1007/s00216-014-7724-9
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发表时间：
2014-05-01
影响因子：
4.3
通讯作者：
Banfi, Cristina
中科院分区：
文献类型：
--
作者：
Brioschi, Maura;Eligini, Sonia;Banfi, Cristina
本文描述了一种微蛋白质组学工作流程，可用于同时鉴定和定量来自最小数量的形态异质培养贴壁细胞的蛋白质。分析策略利用激光捕获显微解剖，一种有效的手段，收获纯细胞群，和无标签质谱。我们优化了工作流程，特别是参考细胞固定，这对于成功的基于激光的显微解剖和下游分子研究至关重要。此外，我们定义了要分离的细胞的最小数量，并分析了令人满意的蛋白质组覆盖率。为了建立这个工作流程，我们选择了体外自发分化的人单核细胞来源的巨噬细胞。在我们的培养条件下，这些细胞表现出不同的形态，使人想起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为培养异质细胞的生物学和病理学提供新的见解，特别是当其他细胞选择方法不合适时。
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.