Biomolecular Mass Spectrometry & Proteomics, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands.
J Am Heart Assoc. 2013 Aug 7;2(4):e000318. doi: 10.1161/JAHA.113.000318.
The multifunctional Ca(2+)- and calmodulin-dependent protein kinase II (CaMKII) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of CaMKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful approach to determining CaMKII's role therein is large-scale analysis of phosphorylation events by mass spectrometry. However, current large-scale phosphoproteomics approaches have proved inadequate for high-fidelity identification of kinase-specific roles. The purpose of this study was to develop a phosphoproteomics approach to specifically identify CaMKII's downstream effects in cardiac tissue.
To identify putative downstream CaMKII targets in cardiac tissue, animals with myocardial-delimited expression of the specific peptide inhibitor of CaMKII (AC3-I) or an inactive control (AC3-C) were compared using quantitative phosphoproteomics. The hearts were isolated after isoproterenol injection to induce CaMKII activation downstream of β-adrenergic receptor agonist stimulation. Enriched phosphopeptides from AC3-I and AC3-C mice were differentially quantified using stable isotope dimethyl labeling, strong cation exchange chromatography and high-resolution LC-MS/MS. Phosphorylation levels of several hundred sites could be profiled, including 39 phosphoproteins noticeably affected by AC3-I-mediated CaMKII inhibition.
Our data set included known CaMKII substrates, as well as several new candidate proteins involved in functions not previously implicated in CaMKII signaling.
多功能钙（Ca 2+ ）和钙调蛋白依赖性蛋白激酶 II（CaMKII）是心脏生理学和病理学的关键介质。CaMKII 的表达和激活增加与心律失常事件的风险升高有关，是人类心力衰竭的标志。通过质谱法大规模分析磷酸化事件是确定 CaMKII 在此过程中作用的一种有用方法。然而，当前的大规模磷酸蛋白质组学方法已被证明不足以高保真地识别激酶特异性作用。本研究的目的是开发一种磷酸蛋白质组学方法，以专门鉴定心脏组织中 CaMKII 的下游效应。
为了鉴定心脏组织中 CaMKII 的潜在下游靶标，使用定量磷酸蛋白质组学比较了心肌局限性表达特定 CaMKII 肽抑制剂（AC3-I）或无活性对照（AC3-C）的动物。用异丙肾上腺素注射诱导心脏组织中 CaMKII 激活，以刺激β-肾上腺素能受体激动剂刺激后的 CaMKII 激活。使用稳定同位素二甲基标记、强阳离子交换色谱和高分辨率 LC-MS/MS 对 AC3-I 和 AC3-C 小鼠的富集磷酸肽进行差异定量。可以分析几百个磷酸化位点的水平，包括 39 个明显受 AC3-I 介导的 CaMKII 抑制影响的磷酸蛋白。
我们的数据集中包括已知的 CaMKII 底物，以及几个以前未涉及 CaMKII 信号传导的新候选蛋白，这些蛋白参与了功能。
