Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
Molecules. 2021 Nov 11;26(22):6816. doi: 10.3390/molecules26226816.
Sample preparation is the most critical step in proteomics as it directly affects the subset of proteins and peptides that can be reliably identified and quantified. Although a variety of efficient and reproducible sample preparation strategies have been developed, their applicability and efficacy depends much on the biological sample. Here, three approaches were evaluated for the human milk and plasma proteomes. Protein extracts were digested either in an ultrafiltration unit (filter-aided sample preparation, FASP) or in-solution (ISD). ISD samples were desalted by solid-phase extraction prior to nRPC-ESI-MS/MS. Additionally, milk and plasma samples were directly digested by FASP without prior protein precipitation. Each strategy provided inherent advantages and disadvantages for milk and plasma. FASP appeared to be the most time efficient procedure with a low miscleavage rate when used for a biological sample aliquot, but quantitation was less reproducible. A prior protein precipitation step improved the quantitation by FASP due to significantly higher peak areas for plasma and a much better reproducibility for milk. Moreover, the miscleavage rate for milk, the identification rate for plasma, and the carbamidomethylation efficiency were improved. In contrast, ISD of both milk and plasma resulted in higher miscleavage rates and is therefore less suitable for targeted proteomics.
样品制备是蛋白质组学中最关键的步骤，因为它直接影响到可以可靠鉴定和定量的蛋白质和肽的子集。尽管已经开发了多种高效且可重复的样品制备策略，但它们的适用性和效果在很大程度上取决于生物样品。在这里，我们评估了三种方法用于人乳和血浆蛋白质组学。蛋白质提取物分别在超滤单元（过滤辅助样品制备，FASP）或溶液中（ISD）进行消化。在进行 nRPC-ESI-MS/MS 之前，ISD 样品通过固相萃取进行脱盐。此外，牛奶和血浆样品可以直接通过 FASP 进行消化，而无需进行蛋白质预沉淀。每种策略都为牛奶和血浆提供了内在的优缺点。当用于生物样品等分试样时，FASP 似乎是最有效率的方法，具有较低的错误切割率，但定量重复性较差。由于蛋白质沉淀前处理步骤显著提高了血浆的峰面积，并且显著提高了牛奶的重复性，因此通过 FASP 进行蛋白质沉淀前处理可以提高定量。此外，牛奶的错误切割率、血浆的鉴定率和碳化二亚胺的效率都得到了提高。相比之下，ISD 对牛奶和血浆都导致了更高的错误切割率，因此不太适合靶向蛋白质组学。
