There is a growing interest for investigating endogenous peptides from human biofluids which may provide yet unknown functional benefits or provide an early indication of disease states as potential biomarkers. A major technical bottleneck in the investigation of endogenous peptides from body fluids, e.g., serum, urine, saliva, and milk, is that each of these fluids seems to require unique workflows for peptide extraction and analysis. Thus, protocols optimized for serum cannot be directly translated to milk. One biofluid that is readily available, but which has not been extensively explored, is human milk, whose analysis could contribute to our understanding of the immune development of the newborn infant. Due to the occurrence of highly abundant lipids, proteins, and saccharides, milk peptidomics requires dedicated sample preparation steps. The aim of this study was to develop a time and cost-efficient workflow for the analysis of the human milk peptidome, for which we compared peptide extraction methodologies and peptide fragmentation methods. A method using strong acid protein precipitation and analysis by collision-induced dissociation fragmentation was found to be superior to all other test methods, allowing us qualitative and quantitative detection of about 4000 endogenous human milk peptides in a total analysis time of just 18 h.

研究来自人类生物流体的内源性肽的兴趣日益浓厚，这些内源性肽可能提供未知的功能性益处或作为潜在的生物标记物提供疾病状态的早期指示。研究体液（例如血清，尿液，唾液和牛奶）中的内源肽的主要技术瓶颈是，每种这些流体似乎都需要独特的工作流程来进行肽的提取和分析。因此，针对血清优化的实验方案无法直接翻译成牛奶。人乳是一种容易获得但尚未得到广泛研究的生物流体，其分析有助于我们对新生婴儿的免疫发展的了解。由于存在高度丰富的脂质，蛋白质和糖类，因此牛奶肽组学需要专门的样品制备步骤。这项研究的目的是开发一种节省时间和成本的工作流程来分析人乳肽组，为此我们比较了肽提取方法和肽片段化方法。发现使用强酸蛋白质沉淀和通过碰撞诱导的解离片段进行分析的方法优于所有其他测试方法，从而使我们能够在仅18 h的总分析时间内定性和定量检测约4000种内源性人乳肽。