Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.
Methods Mol Biol. 2024;2823:129-140. doi: 10.1007/978-1-0716-3922-1_9.
Analyzing the phosphoproteome at nanoscale poses a significant challenge, mainly due to the substantial sample loss from nonspecific surface adsorption during the enrichment of low stoichiometric phosphopeptides. Here, we describe a tandem tip-based phosphoproteomics sample preparation method capable of sequential sample cleanup and enrichment without the need for additional sample transfer, thereby minimizing sample loss. Integration of this method to our recently developed SOP (surfactant-assisted one-pot sample preparation) and iBASIL (improved boosting to amplify signal with isobaric labeling) approaches creates a streamlined workflow, enabling sensitive, high-throughput nanoscale phosphoproteomics measurements.
在纳米尺度上分析磷酸化蛋白质组存在重大挑战，主要是由于在低计量磷酸肽的富集过程中，大量的样品会因非特异性表面吸附而损失。在这里，我们描述了一种基于串联尖端的磷酸化蛋白质组学样品制备方法，该方法能够在无需额外样品转移的情况下进行顺序样品清洗和富集，从而最大限度地减少样品损失。将这种方法与我们最近开发的 SOP（表面活性剂辅助的一锅法样品制备）和 iBASIL（用等压标记增强信号的改进提升）方法相结合，创建了一个简化的工作流程，实现了灵敏、高通量的纳米尺度磷酸化蛋白质组学测量。
