Quantitative phosphoproteomics using acetone-based peptide labeling: method evaluation and application to a cardiac ischemia/reperfusion model.
Quantitative phosphoproteomics using acetone-based peptide labeling: method evaluation and application to a cardiac ischemia/reperfusion model.
复制标题
使用基于丙酮的肽标记进行定量磷酸蛋白质组学：方法评估及其在心脏缺血/再灌注模型中的应用。
DOI：
10.1021/pr400835k
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发表时间：
2013
影响因子：
4.4
通讯作者：
Greis,KennethD
中科院分区：
文献类型：
--
作者：
Wijeratne,ArunaB;Manning,JanetR;Schultz,JoElJ;Greis,KennethD
质谱（MS）技术在与生理或病理变化相关的细胞系统中全面描述蛋白质磷酸化，已成为生物学研究的重要兴趣。一种基于质谱的策略利用廉价的丙酮肽标记技术，称为丙酮还原烷基化（RABA），用于定量磷酸化蛋白质组学，以评估其能力。由于RABA标记磷酸化分析的化学性质之前没有报道，因此首先使用标准磷蛋白和来自心脏组织提取物的相同磷蛋白组进行验证。然后使用工作流来比较不表达FGF2的小鼠心脏组织磷酸化蛋白质组与表达低分子成纤维细胞生长因子-2 （LMW FGF2）的心脏组织磷酸化蛋白质组，以将低分子成纤维细胞生长因子-2 （LMW FGF2）介导的心脏缺血/再灌注损伤诱导的心脏保护现象与LMW FGF2信号级联的下游磷酸化变化联系起来。在10种不同蛋白的14个不同位点上发现了具有统计学意义的磷酸化变化，其中一些具有已经建立的LMW FGF2介导的心脏保护信号机制（例如连接蛋白43），一些具有将LMW FGF2与心脏保护机制联系起来的新细节（例如心肌肌球蛋白结合蛋白C或cMyBPC），以及一些以前未被LMW FGF2识别为心脏保护信号的新的下游效应物。此外，鉴定出的其中一个磷酸肽cMyBPC/pSer-282通过基于SRM（选择反应监测）的方法进行位点特异性定量进一步验证，该方法也依赖于以氘化丙酮作为内标的合成磷酸肽的同位素标记。总体而言，本研究证实，廉价的丙酮肽标记可用于探索性和靶向定量磷酸化蛋白质组学研究，以识别和验证整个组织中生物学相关的磷酸化变化。
Mass spectrometry (MS) techniques to globally profile protein phosphorylation in cellular systems that are relevant to physiological or pathological changes have been of significant interest in biological research. An MS-based strategy utilizing an inexpensive acetone-based peptide-labeling technique known as reductive alkylation by acetone (RABA) for quantitative phosphoproteomics was explored to evaluate its capacity. Because the chemistry for RABA labeling for phosphorylation profiling had not been previously reported, it was first validated using a standard phosphoprotein and identical phosphoproteomes from cardiac tissue extracts. A workflow was then utilized to compare cardiac tissue phosphoproteomes from mouse hearts not expressing FGF2 versus hearts expressing low-molecular-weight fibroblast growth factor-2 (LMW FGF2) to relate low-molecular-weight fibroblast growth factor-2 (LMW FGF2)-mediated cardioprotective phenomena induced by ischemia/reperfusion injury of hearts, with downstream phosphorylation changes in LMW FGF2 signaling cascades. Statistically significant phosphorylation changes were identified at 14 different sites on 10 distinct proteins, including some with mechanisms already established for LMW FGF2-mediated cardioprotective signaling (e.g., connexin-43), some with new details linking LMW FGF2 to the cardioprotective mechanisms (e.g., cardiac myosin binding protein C or cMyBPC), and also several new downstream effectors not previously recognized for cardio-protective signaling by LMW FGF2. Additionally, one of the phosphopeptides, cMyBPC/pSer-282, identified was further verified with site-specific quantification using an SRM (selected reaction monitoring)-based approach that also relies on isotope labeling of a synthetic phosphopeptide with deuterated acetone as an internal standard. Overall, this study confirms that the inexpensive acetone-based peptide labeling can be used in both exploratory and targeted quantification phosphoproteomic studies to identify and verify biologically relevant phosphorylation changes in whole tissues.