本研究选取了ERK1（Pro-定向）、CK2（酸性）和PKA（碱性）这三种具有不同底物选择性的代表性蛋白激酶，用以研究包含来自三个不同细胞系的蛋白质组的底物池中的磷酸化序列模式。具体来说，我们将重组激酶添加到细胞提取的蛋白质组中，使其在体外磷酸化底物。离心酶消化后，富集磷酸肽并进行nanoLC/MS/MS分析，以大规模确定其磷酸化位点。通过分析所得的磷酸化位点及其周围序列，得到了每种激酶-底物质组对应的磷酸化模式。我们发现，每种激酶在不同的底物池蛋白质组中表现出相同的磷酸化模式，与底物池蛋白质组无关。此外，所确定的模式也与完全随机的肽库中发现的模式一致。这些结果表明，细胞提取的蛋白质组可以提供具有足够准确性的激酶磷酸化模式，尽管其序列并非完全随机，从而支持基于细胞提取物的磷蛋白质组学分析作为对于感兴趣的激酶底物池的磷酸化模式鉴定的鲁棒性。 © 2023 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Three representative protein kinases with different substrate preferences, ERK1 (Pro-directed), CK2 (acidophilic), and PKA (basophilic), were used to investigate phosphorylation sequence motifs in substrate pools consisting of the proteomes from three different cell lines, MCF7 (human mammary carcinoma), HeLa (human cervical carcinoma), and Jurkat (human acute T-cell leukemia). Specifically, recombinant kinases were added to the cell-extracted proteomes to phosphorylate the substrates in vitro. After trypsin digestion, the phosphopeptides were enriched and subjected to nanoLC/MS/MS analysis to identify their phosphorylation sites on a large scale. By analyzing the obtained phosphorylation sites and their surrounding sequences, phosphorylation motifs were extracted for each kinase-substrate proteome pair. We found that each kinase exhibited the same set of phosphorylation motifs, independently of the substrate pool proteome. Furthermore, the identified motifs were also consistent with those found using a completely randomized peptide library. These results indicate that cell-extracted proteomes can provide kinase phosphorylation motifs with sufficient accuracy, even though their sequences are not completely random, supporting the robustness of phosphorylation motif identification based on phosphoproteome analysis of cell extracts as a substrate pool for a kinase of interest.© 2023 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.