Motif-targeting phosphoproteome analysis of cancer cells for profiling kinase inhibitors.

We developed a motif-enriched phosphoproteome analysis workflow utilizing an in vitro kinase reaction to enrich a set of peptides with specific primary sequence motifs. The kinase reaction attached the phosphate tag to the peptides derived from phosphopeptide enrichment and subsequent dephosphorylation, facilitates the enrichment of the kinase substrates from phosphoproteomic samples. We applied this method to phosphoproteomic profiling of inhibitor treated cancer cells, and successfully profiled kinase inhibitors with different inhibitory spectra. We anticipate this workflow will be useful to target a specific set of the phosphoproteome with the wide variety of available recombinant protein kinases.

我们开发了一种基于体外激酶反应的基序富集型磷酸化蛋白质组分析流程，可用于富集带有特异性一级序列基序的肽段。该激酶反应将磷酸基团标签连接至经磷酸肽富集及后续去磷酸化得到的肽段，从而助力从磷酸化蛋白质组样品中富集激酶底物。我们将该方法应用于经抑制剂处理的癌细胞的磷酸化蛋白质组谱分析，成功表征了具有不同抑制谱的激酶抑制剂。我们预期，借助种类丰富的重组蛋白激酶，该流程可用于靶向特定范围的磷酸化蛋白质组。