Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan.
Department of Frontier Research and Development, Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan.
Int J Mol Sci. 2021 Mar 4;22(5):2565. doi: 10.3390/ijms22052565.
A conditioned medium of a cell culture is widely used for various biological applications and frequently analyzed to characterize the functional proteins responsible for observed biological functions. However, a large number of abundant proteins in fetal bovine serum (FBS), usually included in the conditioned medium of a mammalian cell culture medium, hampers in-depth proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For a deep proteomic analysis of a conditioned medium by LC-MS/MS, we developed a simple albumin depletion approach coupled with data-independent acquisition (DIA)-mode LC-MS/MS for the conditioned medium of mammalian cells in this study. The results showed that this approach enabled the detection of more than 3700 cell-derived proteins in the cell culture supernatant containing FBS. We further demonstrated the potency of this approach by analyzing proteins in the conditioned media of HeLa cells with and without tumor necrosis factor (TNF) stimulation: >40 differentially accumulated proteins, including four cytokines, upon TNF stimulation were identified in the culture media, which were hardly detected by conventional proteome approaches in the literature.
细胞培养的条件培养基广泛应用于各种生物学应用，并经常进行分析以鉴定负责观察到的生物学功能的功能蛋白。然而，胎牛血清（FBS）中的大量丰富蛋白通常包含在哺乳动物细胞培养基的条件培养基中，这会阻碍通过液相色谱-串联质谱（LC-MS/MS）进行深入的蛋白质组学分析。为了通过 LC-MS/MS 对条件培养基进行深入的蛋白质组学分析，我们在本研究中开发了一种简单的白蛋白去除方法，该方法与数据非依赖性采集（DIA）模式 LC-MS/MS 相结合，用于哺乳动物细胞的条件培养基。结果表明，该方法能够在含有 FBS 的细胞培养上清液中检测到超过 3700 种细胞来源的蛋白质。我们通过分析有和没有肿瘤坏死因子（TNF）刺激的 HeLa 细胞条件培养基中的蛋白质进一步证明了这种方法的有效性：在培养基中鉴定出 >40 种差异积累的蛋白质，包括四种细胞因子，而在文献中的常规蛋白质组学方法中几乎检测不到这些蛋白质。
