Secretome analysis is broadly applied to understand the interplay between cells and their microenvironment. In particular, the unbiased analysis by mass spectrometry‐based proteomics of conditioned medium has been successfully applied. In this context, several approaches have been developed allowing to distinguish proteins actively secreted by cells from proteins derived from culture medium or proteins released from dying cells. Here, three different methods comparing conditioned medium and lysate by quantitative mass spectrometry‐based proteomics to identify bona fide secreted proteins are evaluated. Evaluation in three different human cell lines reveals that all three methods give access to a similar set of bona fide secreted proteins covering a broad abundance range. In the analyzed primary cells, that is, mesenchymal stromal cells and normal human dermal fibroblasts, more than 70% of the identified proteins are linked to classical secretion pathways. Furthermore, 4–12% are predicted to be released by unconventional secretion pathways. Interestingly, evidence of release by ectodomain shedding in a large number of the remaining candidate proteins is found. In summary, it is convinced that comparative secretomics is currently the method of choice to obtain high‐confident secretome data and to identify novel candidates for unconventional protein secretion which have been neglected so far.

中文翻译：

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比较 Secretomics 可获取高度可信的分泌组数据：评估确定真正分泌蛋白的不同方法

分泌组分析被广泛应用于了解细胞与其微环境之间的相互作用。特别是，基于质谱的条件培养基蛋白质组学的无偏分析已得到成功应用。在这种情况下，已经开发了几种方法来区分细胞主动分泌的蛋白质与来自培养基的蛋白质或从垂死细胞释放的蛋白质。在这里，评估了通过基于定量质谱的蛋白质组学比较条件培养基和裂解物以鉴定真正分泌的蛋白质的三种不同方法。在三种不同的人类细胞系中的评估表明，所有三种方法都可以获得一组相似的真正分泌的蛋白质，涵盖了广泛的丰度范围。在分析的原代细胞中，即 在间充质基质细胞和正常人皮肤成纤维细胞中，超过 70% 的鉴定蛋白质与经典分泌途径有关。此外，预计 4-12% 将通过非常规分泌途径释放。有趣的是，发现了大量剩余候选蛋白质通过胞外域脱落而释放的证据。总之，确信比较分泌组学是目前获得高可信分泌组数据和鉴定迄今为止被忽视的非常规蛋白质分泌的新候选者的首选方法。发现了在大量剩余候选蛋白质中通过胞外域脱落而释放的证据。总之，确信比较分泌组学是目前获得高可信分泌组数据和鉴定迄今为止被忽视的非常规蛋白质分泌的新候选者的首选方法。发现了在大量剩余候选蛋白质中通过胞外域脱落而释放的证据。总之，确信比较分泌组学是目前获得高可信分泌组数据和鉴定迄今为止被忽视的非常规蛋白质分泌的新候选者的首选方法。