Reversible phosphorylation plays critical roles in cell growth, division, and signal transduction. Kinases which catalyze the transfer of γ-phosphate groups of nucleotide triphosphates to their substrates are central to the regulation of protein phosphorylation and are therefore important therapeutic targets. Top-down mass spectrometry (MS) presents unique opportunities to study protein kinases owing to its capabilities in comprehensive characterization of proteoforms that arise from alternative splicing, sequence variations, and post-translational modifications. Here, for the first time, we developed a top-down MS method to characterize the catalytic subunit (C-subunit) of an important kinase, cAMP-dependent protein kinase (PKA). The recombinant PKA C-subunit was expressed in Escherichia coli and successfully purified via his-tag affinity purification. By intact mass analysis with high resolution and high accuracy, four different proteoforms of the affinity-purified PKA C-subunit were detected, and the most abundant proteoform was found containing seven phosphorylations with the removal of N-terminal methionine. Subsequently, the seven phosphorylation sites of the most abundant PKA C-subunit proteoform were characterized simultaneously using tandem MS methods. Four sites were unambiguously identified as Ser10, Ser11, Ser18, and Ser30, and the remaining phosphorylation sites were localized to Ser2/Ser3, Ser358/Thr368, and Thr[215-224]Tyr in the PKA C-subunit sequence with a 20mer 6xHis-tag added at the N-terminus. Interestingly, four of these seven phosphorylation sites were located at the 6xHis-tag. Furthermore, we have performed dephosphorylation reaction by Lambda protein phosphatase and showed that all phosphorylations of the recombinant PKA C-subunit phosphoproteoforms were removed by this phosphatase.

中文翻译：

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通过自上而下质谱法对 cAMP 依赖性蛋白激酶的重组催化亚基进行综合表征。

可逆磷酸化在细胞生长、分裂和信号转导中起着关键作用。催化三磷酸核苷酸的γ-磷酸基团转移到其底物的激酶是调节蛋白质磷酸化的核心，因此是重要的治疗靶点。自上而下的质谱 (MS) 提供了研究蛋白激酶的独特机会，因为它能够全面表征由可变剪接、序列变异和翻译后修饰产生的蛋白质型。在这里，我们首次开发了一种自上而下的 MS 方法来表征重要激酶 cAMP 依赖性蛋白激酶 (PKA) 的催化亚基 (C 亚基)。重组 PKA C 亚基在大肠杆菌中表达，并通过 His 标签亲和纯化成功纯化。通过高分辨率和高精度的完整质量分析，检测到亲和纯化的 PKA C 亚基的四种不同蛋白型，发现最丰富的蛋白型含有 7 种磷酸化，并去除了 N 端甲硫氨酸。随后，使用串联质谱法同时表征了最丰富的 PKA C 亚基蛋白型的七个磷酸化位点。四个位点被明确鉴定为 Ser10、Ser11、Ser18 和 Ser30，其余磷酸化位点位于 PKA C 亚基序列中的 Ser2/Ser3、Ser358/Thr368 和 Thr[215-224]Tyr，具有 20mer 6xHis - 在 N 端添加的标签。有趣的是，这七个磷酸化位点中有四个位于 6xHis 标签。此外，我们已经通过 Lambda 蛋白磷酸酶进行了去磷酸化反应，并表明该磷酸酶去除了重组 PKA C 亚基磷酸化蛋白型的所有磷酸化。