Institute of Molecular Medicine I, Proteome Research, University Hospital Düsseldorf, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Methods Mol Biol. 2021;2228:293-306. doi: 10.1007/978-1-0716-1024-4_21.
Cells secrete proteins to communicate with their environment. Therefore, it is interesting to characterize the proteins which are released from cells under certain experimental conditions the so-called secretome. Here, often proteins from conditioned medium of cultured cells are analyzed, but these additionally might include also contaminating proteins of serum that have not been sufficiently removed or proteins from dying cells. To provide high-quality secretome data and minimize potential contaminants, we describe a quantitative comparison of conditioned medium and the cellular proteome. The described workflow comprises cell cultivation, sample preparation, and final data analysis which is based on the comparison of data from label-free mass spectrometric quantification of proteins from the conditioned medium with corresponding cellular proteomes enabling the detection of bona fide secreted proteins.
细胞通过分泌蛋白质与周围环境进行交流。因此，对特定实验条件下从细胞中释放的蛋白质（即分泌组）进行特征分析是很有意义的。这里，通常分析的是培养细胞的条件培养基中的蛋白质，但这些蛋白质还可能包括尚未充分去除的血清中的污染蛋白或来自死亡细胞的蛋白。为了提供高质量的分泌组数据并最大限度地减少潜在的污染物，我们描述了条件培养基和细胞蛋白质组的定量比较。所描述的工作流程包括细胞培养、样品制备和最终数据分析，该分析基于对来自条件培养基的蛋白质进行无标记质谱定量的数据与相应的细胞蛋白质组数据的比较，从而可以检测真正分泌的蛋白质。
