We show that parallel reaction monitoring (PRM) can be used for exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled peptides. We have compared two different PRM approaches on a digest of a U87 cell culture, namely, direct-PRM (tryptic digest measured by PRM without any further sample preparation) and TiO₂-PRM (tryptic digest enriched with TiO₂ cartridges, followed by PRM measurement); these approaches are compared for the following phosphorylation sites: neuroblast differentiation-associated protein (AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor (EGFR S1166-p). A reproducible percentage of phosphorylation could be determined (CV 6–13%) using direct-PRM or TiO₂-PRM. In addition, we tested the approaches in a cell culture experiment in which U87 cells were deprived of serum. As a “gold standard” we included immune precipitation of EGFR followed by PRM (IP-PRM). For EGFR (S1166) and AHNAK (S5480) a statistical significant change in the percentage of phosphorylation could be observed as a result of serum deprivation; for EGFR (S1166) this change was observed for both TiO₂-PRM and IP-PRM. The presented approach has the potential to multiplex and to quantify the ratio of phosphorylation in a single analysis.

中文翻译：

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靶向质谱法测定蛋白质中特定位点的磷酸化率

我们表明，平行反应监测（PRM）可用于使用稳定同位素标记的肽对蛋白质的磷酸化率进行精确定量。我们比较两个不同的PRM上的一个U87细胞培养物的消化，即直接-PRM方法（胰蛋白酶消化通过PRM测量而无需任何进一步的样品制备）和TiO ₂ -PRM（胰蛋白酶消化富含的TiO ₂的墨盒，接着PRM测量）; 对以下磷酸化位点比较了这些方法：成神经细胞分化相关蛋白（AHNAK S5480-p），钙/钙调蛋白依赖性蛋白激酶II型亚基δ（CAMK2D T337-p）和表皮生长因子受体（EGFR S1166-p ）。使用直接PRM或TiO可以测定可再现的磷酸化百分比（CV 6–13％）₂ -PRM。另外，我们在细胞培养实验中测试了其中的方法，其中U87细胞被剥夺了血清。作为“黄金标准”，我们包括EGFR的免疫沉淀，然后是PRM（IP-PRM）。对于EGFR（S1166）和AHNAK（S5480），由于血清剥夺，可观察到磷酸化百分比的统计学显着变化。对于EGFR（S1166），TiO ₂ -PRM和IP-PRM均观察到这种变化。所提出的方法具有在单个分析中多路复用和量化磷酸化比例的潜力。