The Fifth People's Hospital of Shanghai, Shanghai Key Laboratory of Medical Epigenetics, The International Co-Laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.
Wuhan National Laboratory for Optoelectronics, MoE Key Laboratory for Biomedical Photonics, School of Engineering Sciences, Huazhong University of Science and Technology, Wuhan 430074, China.
Anal Chem. 2023 Jul 18;95(28):10703-10712. doi: 10.1021/acs.analchem.3c01486. Epub 2023 Jul 5.
Recent developments in phosphoproteomics have enabled signaling studies where over 10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in sample size, reproducibility, and robustness, hampering experiments that involve low-input samples such as rare cells and fine-needle aspiration biopsies. To address these challenges, we introduced a simple and rapid phosphorylation enrichment method (miniPhos) that uses a minimal amount of the sample to get enough information to decipher biological significance. The miniPhos approach completed the sample pretreatment within 4 h and high effectively collected the phosphopeptides in a single-enrichment format with an optimized enrichment process and miniaturized system. This resulted in an average of 22,000 phosphorylation peptides quantified from 100 μg of proteins and even confidently localized over 4500 phosphosites from as little as 10 μg of peptides. Further application was carried out on different layers of mouse brain micro-sections; our miniPhos method provided quantitative information on protein abundance and phosphosite regulation for the most relevant neurodegenerative diseases, cancers, and signaling pathways in the mouse brain. Surprisingly, the phosphoproteome exhibited more spatial variations than the proteome in the mouse brain. Overall, spatial dynamics of phosphosites are integrated with proteins to gain insights into crosstalk of cellular regulation at different layers, thereby facilitating a more comprehensive understanding of mouse brain development and activity.
磷酸化蛋白质组学的最新进展使得信号研究得以实现，其中超过 10000 个磷酸化位点可以被常规识别和定量。然而，目前的分析在样本量、可重复性和稳健性方面受到限制，阻碍了涉及低输入样本（如稀有细胞和细针穿刺活检）的实验。为了解决这些挑战，我们引入了一种简单快速的磷酸化富集方法（miniPhos），该方法仅使用少量样本即可获得足够的信息来破译生物学意义。miniPhos 方法在 4 小时内完成样品预处理，并通过优化的富集过程和小型化系统以单一富集的形式高效地收集磷酸肽。这使得从 100 μg 蛋白质中定量到 22000 个磷酸肽，甚至从 10 μg 肽中就能准确地定位 4500 多个磷酸化位点。进一步在不同层的小鼠脑微切片上进行了应用；我们的 miniPhos 方法为与最相关的神经退行性疾病、癌症和信号通路相关的蛋白质丰度和磷酸化位点调节提供了定量信息。令人惊讶的是，磷酸化蛋白质组在小鼠脑中比蛋白质组表现出更多的空间变化。总的来说，磷酸化位点的空间动态与蛋白质相结合，深入了解不同层细胞调节的串扰，从而促进对小鼠脑发育和活动的更全面理解。
