Department of Cancer Biology and ‡Department of Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine , 3125 Eden Avenue,Cincinnati, Ohio 45267, United States.
J Proteome Res. 2013 Oct 4;12(10):4268-79. doi: 10.1021/pr400835k. Epub 2013 Sep 24.
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.
质谱（MS）技术可广泛分析与生理或病理变化相关的细胞系统中的蛋白质磷酸化，这在生物研究中具有重要意义。本研究探索了一种基于 MS 的策略，该策略利用一种廉价的丙酮基肽标记技术（称为丙酮还原烷基化（RABA））进行定量磷酸蛋白质组学研究，以评估其能力。由于之前未报道过用于磷酸化谱分析的 RABA 标记化学，因此首先使用标准磷酸蛋白和来自心脏组织提取物的相同磷酸蛋白质组对其进行了验证。然后，利用工作流程比较了不表达 FGF2 的小鼠心脏与表达低分子量成纤维细胞生长因子-2（LMW FGF2）的心脏的心脏组织磷酸蛋白质组，以将低分子量成纤维细胞生长因子-2（LMW FGF2）介导的缺血/再灌注损伤引起的心脏保护现象与 LMW FGF2 信号级联中的下游磷酸化变化联系起来。在 10 个不同蛋白质的 14 个不同位点上鉴定出具有统计学意义的磷酸化变化，其中一些机制已经确立为 LMW FGF2 介导的心脏保护信号（例如，连接蛋白-43），一些新的细节将 LMW FGF2 与心脏保护机制联系起来（例如，肌球蛋白结合蛋白 C 或 cMyBPC），还有一些之前未被认为是 LMW FGF2 心脏保护信号的新下游效应物。此外，鉴定出的一种磷酸肽 cMyBPC/pSer-282 还使用基于 SRM（选择反应监测）的方法进行了位点特异性定量，该方法还依赖于用氘代丙酮对合成磷酸肽进行同位素标记作为内部标准。总的来说，这项研究证实，廉价的丙酮基肽标记可用于探索性和靶向定量磷酸蛋白质组学研究，以识别和验证整个组织中具有生物学意义的磷酸化变化。
