Proteomics Group, Department of System Biology, Biomedical Research Division, Center for Genetic Engineering& Biotechnology (CIGB), Havana 10600, Cuba.
Department of Animal and Human Biology, Faculty of Biology, University of Havana, Havana 10600, Cuba; Molecular Oncology Group, Department of Pharmaceuticals, Biomedical Research Division, Center for Genetic Engineering & Biotechnology (CIGB), Havana 10600, Cuba.
J Proteomics. 2024 Sep 15;307:105269. doi: 10.1016/j.jprot.2024.105269. Epub 2024 Aug 2.
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.
定量磷酸化蛋白质组学数据主要是通过比较两种或更多不同条件下相对磷酸肽强度的实验来报告的，而理想的比较参数是磷酸肽占有率。这个术语很少被使用，因此在磷酸蛋白质组学研究中几乎没有被实施，这应该引起科学期刊的关注。为了证明这个问题的相关性，我们展示了选择的方法如何影响数据的解释。这里显示了在 CK2 抑制后，两种 AML 细胞系中的磷酸蛋白质组谱发生变化的情况。在 CK2 抑制后，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。即使使用强度计算方法可以检测到更多的定量磷酸肽，在所有情况下，对 CK2 抑制反应下调的 CK2 共识序列的百分比都更高，这是使用磷酸位点占有率定量的结果。值得注意的是，发现大量的 CK2 共识序列下调，磷酸位点占有率变化至少为 10%或 15%，这表明低占有率调节阈值可能是生物效应的指示。此外，只有在通过占有率定量的磷酸蛋白质组中，几个生物学过程才会显著过表达。占有率变化范围内的功能富集分析也说明了受到 CX-4945 抑制的 AML 细胞系之间的明显差异。通过强度和占有率定量的磷酸蛋白质组之间的重叠很低，这表明需要新的蛋白质组学技术发展来提高占有率方法的性能。即使在这种情况下，结果表明占有率定量比基于强度的磷酸化定量表现更好，这加强了占有率定量方法对描述磷酸蛋白质组数据的重要性。
