Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA.
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.
Commun Biol. 2023 Jan 18;6(1):70. doi: 10.1038/s42003-022-04400-x.
Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We develop a tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3000 (>9500) phosphopeptides from 1 (10) µg of cell lysate using the label-free method without a spectral library. It also enables precise quantification of ~600 phosphopeptides from 100 sorted cells (single-cell level input for the enriched phosphopeptides) and ~700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 µm (equivalent to ~100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.
纳米级样品分析的有效磷酸化蛋白质组学仍然是一项艰巨的任务，主要是由于在低计量磷酸肽的富集过程中，与非特异性表面吸附相关的大量样品损失。我们开发了一种串联尖端磷酸化蛋白质组学样品制备方法，能够在不进行额外样品转移的情况下进行样品清洗和富集，并且将其与我们最近开发的 SOP（表面活性剂辅助的单步样品制备）和 iBASIL（改进的同量异位标记信号放大）方法相结合，提供了一种简化的工作流程，能够实现灵敏、高通量的纳米级磷酸化蛋白质组学测量。该方法显著减少了样品损失和处理时间，使用无标记方法，从 1（10）µg 的细胞裂解物中鉴定出超过 3000（9500）个磷酸肽，而无需谱库。它还能够从 100 个分选细胞（富集磷酸肽的单细胞水平输入）中精确定量约 600 个磷酸肽，从人类脾脏组织体素中精确定量约 700 个磷酸肽，体素空间分辨率为 200µm（相当于约 100 个细胞），具有高通量的特点。新的工作流程为在低纳克水平对质量有限的样品进行磷酸化蛋白质组学分析开辟了途径。
