Recently, we reported a fast on-line alkaline micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric approach for sensitive phosphopeptide screening of a tryptic digested protein and subsequent characterization of the identified phosphopeptide. Based on this study, we now applied an improved method for the identification of phosphorylation sites in insulin receptor substrate 1, an important mediator in insulin signal transduction which was phosphorylated in vitro by protein kinase C-zeta. The approach consists of an on-line alkaline negative-ion micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric hybrid scan experiment using a triple-quadrupole mass spectrometer with fractionation and subsequent off-line nanoES-MS (ion trap) analysis of the phosphopeptide-containing fractions. During the liquid chromatography (LC)/ES-MS experiment, the phosphopeptides of the enzymatic digest mixture of the studied insulin receptor substrate 1 fragment were detected under high skimmer potential (API-CID) using phosphorylation-specific m/z 79 marker ions as well as the intact m/z-values of the peptides which were recorded under low skimmer potential. Subsequently, the targeted fractions were analyzed by off-line nanoES-MS/MS and MS(3). Using this approach, serine 318 was clearly identified as a major in vitro protein kinase C-zeta phosphorylation site in the insulin receptor substrate -1 fragment. Together, our results indicate that the applied strategy is useful for unequivocal and fast analysis of phosphorylation sites in low abundant signaling proteins.

中文翻译：

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通过负离子muLC / ES-API-CID-MS混合扫描技术鉴定体外胰岛素受体底物1磷酸化位点。

最近，我们报道了一种快速在线碱性微液相色谱/电喷雾-大气电离/碰撞诱导的解离/质谱法，用于胰蛋白酶消化的蛋白质的敏感磷酸肽筛选和随后鉴定的磷酸肽的表征。基于这项研究，我们现在应用一种改进的方法来鉴定胰岛素受体底物1中的磷酸化位点，该底物是胰岛素信号转导的重要介体，在体外被蛋白激酶C-zeta磷酸化。该方法包括在线碱性负离子微液相色谱/电喷雾-大气电离/碰撞诱导解离/质谱混合扫描实验，使用三重四极杆质谱仪进行分馏并随后进行离线nanoES-MS （离子阱）分析含磷酸肽的馏分。在液相色谱（LC）/ ES-MS实验过程中，使用磷酸化特异性m / z 79标记离子作为高分离器电势（API-CID），检测了研究的胰岛素受体底物1片段的酶消化混合物的磷酸肽。以及在低撇渣器电位下记录的完整的m / z值。随后，通过离线nanoES-MS / MS和MS（3）分析了目标馏分。使用这种方法，丝氨酸318被明确确定为胰岛素受体底物-1片段中的主要体外蛋白激酶C-zeta磷酸化位点。总之，我们的结果表明，所应用的策略可用于明确，快速地分析低丰度信号蛋白中的磷酸化位点。