Abstract：
Quantitative phosphoproteomic data has mostly been reported from experiments comparing relative phosphopeptides intensities in two or more different conditions, while the ideal parameter to compare is phosphopeptides occupancies. This term is scarcely used and therefore barely implemented in phosphoproteomics studies, and this should be of concern for the scientific journals. In order to demonstrate the relevance of this issue, here we show how the method of choice affects the interpretation of the data. The phosphoproteomic profile modulated in two AML cell lines after CK2 inhibition with CIGB-300 or CX-4945 is shown. Following the downstream action of CK2 the phosphosite intensity and occupancy results were compared to validate the best approach for quantitative phosphoproteomic studies. Even when the total number of quantified phosphopeptides was higher by using the intensity calculation, in all the cases the percent of CK2 consensus sequences which were down-regulated in response to CK2 inhibition was higher using the phosphosite occupancy quantification. To note, a high number of CK2 consensus sequences was found down-regulated with at least a 10% or 15% of phosphosite occupancy variation illustrating that low thresholds of occupancy modulation might be indicative of biological effect. Additionally, several biological processes only appear significantly over-represented in the phosphoproteome quantified by occupancy. The functional enrichment analysis per ranges of occupancy variations also illustrated clear differences among AML cell lines subjected to CK2 inhibition by CX-4945. A low overlap between the phosphoproteomes quantified by intensity and occupancy was obtained illustrating that new developments in proteomics techniques are needed to improve the performance of the occupancy approach. Even in such context, results indicate that occupancy quantification performs better than phosphorylation quantification based on intensity reinforcing the importance of such quantification approach to describe phosphoproteomic data.
摘要：
定量磷酸蛋白质组数据主要来自比较两种或多种不同条件下相对磷酸肽强度的实验。而比较的理想参数是磷酸肽占用率。该术语很少使用，因此在磷酸化蛋白质组学研究中几乎没有实现。这应该引起科学期刊的关注。为了证明这个问题的相关性，在这里，我们展示了选择方法如何影响数据的解释。显示了用C1GB-300或CX-4945抑制CK2后在两种AML细胞系中调节的磷酸蛋白质组概况。在CK2的下游作用之后,比较磷酸位点强度和占有率结果以验证定量磷酸蛋白质组学研究的最佳方法。即使使用强度计算定量的磷酸肽的总数较高，在所有情况下,响应于CK2抑制而下调的CK2共有序列的百分比使用磷酸化位点占据定量较高。要注意，发现大量CK2共有序列下调,至少10%或15%的磷酸化位点占据变异,说明低阈值的占据调节可能指示生物学效应.此外,几种生物学过程仅在通过占用率定量的磷酸化蛋白质组中出现明显的过度代表。每个占有率变化范围的功能富集分析也说明了受CX-4945抑制CK2的AML细胞系之间的明显差异。获得了通过强度和占有率定量的磷酸蛋白质组之间的低重叠，这表明需要蛋白质组学技术的新发展来改善占有率方法的性能。即使在这样的背景下,结果表明，基于强度增强这种定量方法描述磷酸化蛋白质组数据的重要性，占用定量比磷酸化定量表现更好。
