López Elena, López Isabel, Sequí Julia, Ferreira Antonio
Phosphoproteomic core, Spanish National Cancer Research Centre (CNIO), C/Melchor Fernández Almagro, 3, 28029, Madrid, Spain.
J Clin Bioinforma. 2011 Jul 6;1(1):16. doi: 10.1186/2043-9113-1-16.
The mitogen activated protein kinase (MAPK) pathways are known to be deregulated in many human malignancies. Phosphopeptide identification of protein-kinases and site determination are major challenges in biomedical mass spectrometry (MS). P38 and HuR protein kinases have been reported extensively in the general principles of signalling pathways modulated by phosphorylation, mainly by molecular biology and western blotting techniques. Thus, although it has been demonstrated they are phosphorylated in different stress/stimuli conditions, the phosphopeptides and specific amino acids in which the phosphate groups are located in those protein kinases have not been shown completely.
We have combined different resins: (a) IMAC (Immobilized Metal Affinity Capture), (b) TiO2 (Titanium dioxide) and (c) SIMAC (Sequential Elution from IMAC) to isolate phosphopeptides from p38 and HuR protein kinases in vitro.Different phosphopeptide MS strategies were carried out by the LTQ ion Trap mass spectrometer (Thermo): (a) Multistage activation (MSA) and (b) Neutral loss MS3 (DDNLMS3).In addition, Molecular Dynamics (MD) bioinformatic simulation has been applied in order to simulate, over a period of time, the effects of the presence of the extra phosphate group (and the associated negative charge) in the overall structure and behaviour of the protein HuR.This study is supported by the Declaration of Helsinki and subsequent ethical guidelines.
The combination of these techniques allowed for:(1) The identification of 6 unknown phosphopeptides of these protein kinases. (2) Amino acid site assignments of the phosphate groups from each identified phosphopeptide, including manual validation by inspection of all the spectra. (3) The analyses of the phosphopeptides discovered were carried out in four triplicate experiments to avoid false positives getting high reproducibility in all the isolated phosphopeptides recovered from both protein kinases. (4) Computer simulation using MD techniques allowed us to get functional models of both structure and interactions of the previously mentioned phosphorylated kinases and the differences between their phosphorylated and un-phosphorylated forms.
Many research studies are necessary to unfold the whole signalling network (human proteome), which is so important to advance in clinical research, especially in the cases of malignant diseases.
已知丝裂原活化蛋白激酶（MAPK）通路在许多人类恶性肿瘤中失调。蛋白质激酶的磷酸肽鉴定和位点确定是生物医学质谱（MS）中的主要挑战。P38和HuR蛋白激酶在磷酸化调节的信号通路的一般原理方面已有广泛报道，主要采用分子生物学和蛋白质印迹技术。因此，尽管已证明它们在不同的应激/刺激条件下会发生磷酸化，但这些蛋白激酶中磷酸基团所在的磷酸肽和特定氨基酸尚未完全明确。
我们结合了不同的树脂：（a）固定化金属亲和捕获（IMAC），（b）二氧化钛（TiO2）和（c）IMAC顺序洗脱（SIMAC），以体外分离p38和HuR蛋白激酶的磷酸肽。通过LTQ离子阱质谱仪（赛默飞世尔）进行不同的磷酸肽MS策略：（a）多级活化（MSA）和（b）中性丢失MS3（DDNLMS3）。此外，应用分子动力学（MD）生物信息学模拟，在一段时间内模拟额外磷酸基团（以及相关负电荷）的存在对HuR蛋白整体结构和行为的影响。本研究得到了《赫尔辛基宣言》及后续伦理准则的支持。
这些技术的结合实现了：（1）鉴定出这些蛋白激酶的6种未知磷酸肽。（2）对每个鉴定出的磷酸肽的磷酸基团进行氨基酸位点分配，包括通过检查所有光谱进行人工验证。（3）对发现的磷酸肽进行了四次重复实验分析，以避免假阳性，使从两种蛋白激酶中回收的所有分离磷酸肽都具有高重现性。（4）使用MD技术的计算机模拟使我们能够获得上述磷酸化激酶的结构和相互作用的功能模型，以及它们磷酸化和未磷酸化形式之间的差异。
需要进行许多研究来揭示整个信号网络（人类蛋白质组），这对于推进临床研究，特别是在恶性疾病的情况下非常重要。
