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Updated: May 29, 2026
Oligopeptide Competition Assay for Phosphorylation Site Determination
Published on: May 18, 2017
Discovering and validating unknown phospho-sites from p38 and HuR protein kinases in vitro by Phosphoproteomic and
Elena López1, Isabel López, Julia Sequí
1Phosphoproteomic core, Spanish National Cancer Research Centre (CNIO), C/Melchor Fernández Almagro, 3, 28029, Madrid, Spain. elena.lopez.villar@gmail.com.
Background:
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.
Methods:
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.
Results:
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.
Conclusion:
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.
Insights
This study identifies novel phosphopeptides and phosphorylation sites on p38 and HuR protein kinases using advanced mass spectrometry and bioinformatics. These findings enhance our understanding of kinase signaling in human malignancies.
Area of Science:
- Biochemistry and Molecular Biology
- Proteomics and Mass Spectrometry
- Bioinformatics and Computational Biology
Background:
- Mitogen-activated protein kinase (MAPK) pathways are frequently dysregulated in human cancers.
- Identifying specific phosphopeptides and phosphorylation sites on protein kinases is crucial but challenging for biomedical mass spectrometry (MS).
- Previous studies on p38 and HuR protein kinases have lacked complete phosphosite information despite their known roles in signaling.
Purpose of the Study:
- To identify and characterize novel phosphopeptides and their phosphorylation sites on p38 and HuR protein kinases.
- To investigate the structural and functional impact of phosphorylation on the HuR protein using molecular dynamics simulations.
- To improve phosphoproteomic analysis strategies for complex biological samples.
Main Methods:
- Combined Immobilized Metal Affinity Capture (IMAC), Titanium dioxide (TiO2), and Sequential Elution from IMAC (SIMAC) for phosphopeptide enrichment.
- Employed Multistage Activation (MSA) and Neutral Loss MS3 (DDNLMS3) strategies using an LTQ ion trap mass spectrometer for phosphopeptide analysis.
- Utilized Molecular Dynamics (MD) bioinformatic simulations to model the effects of phosphorylation on the HuR protein structure and behavior.
Main Results:
- Successfully identified six previously unknown phosphopeptides for p38 and HuR protein kinases.
- Determined the specific amino acid sites of phosphorylation for each identified phosphopeptide, with manual spectral validation.
- Achieved high reproducibility through triplicate experiments and generated functional models of phosphorylated vs. unphosphorylated kinases via MD simulations.
Conclusions:
- The integrated phosphoproteomic approach effectively identified novel phosphorylation sites on key protein kinases.
- Molecular dynamics simulations provide insights into the structural consequences of protein phosphorylation.
- Further research into the human phosphoproteome is essential for advancing clinical research, particularly in oncology.
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