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Published on: August 2, 2018
Proteomics pipeline for phosphoenrichment and its application on a human melanoma cell model
Francesco Finamore1, Nadia Ucciferri1, Giovanni Signore2
1Institute of Clinical Physiology, CNR, via Moruzzi 1, Pisa, 56124, Italy.
Abstract:
Cell signalling is tightly regulated by post-translational modification of proteins. Among them, phosphorylation is one of the most interesting and important. Identifying phosphorylation sites on proteins is challenging and requires strategies for pre-separation and enrichment of the phosphorylated species. We applied four different methods for phospho-enrichment involving TiO2 and IMAC matrix to human melanoma cell lysates of starved A375 induced for 1 h with 1% FBS. Comparison of protocol efficiency was evaluated through peptide concentration, sulphur and phosphorus content and peptide analysis by LC-MS in the collected fractions. Our results underlined that each single method is not sufficient for a comprehensive phosphoproteome analysis. In fact, each methodology permits to identify only a fraction of the phosphoproteome contained in a whole cell lysate. The selection of the most efficient protocols and a combination of two phospho-enrichment methods allowed the assessment of this workflow able to pinpoint the main actors in the phospho-proteome cascade of A375 human melanoma cells treated with Vemurafenib.
Insights
Identifying protein phosphorylation sites is crucial for understanding cell signaling. This study compared four phospho-enrichment methods, finding that combining two methods best captures the phosphoproteome in melanoma cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Cell signaling pathways heavily rely on post-translational protein modifications, with phosphorylation being a key regulatory mechanism.
- Accurate identification of phosphorylation sites is essential but technically challenging, necessitating effective enrichment strategies for phosphorylated proteins.
- Human melanoma cells (A375) are a relevant model for studying cancer signaling.
Purpose of the Study:
- To evaluate and compare the efficiency of four distinct phospho-enrichment techniques using titanium dioxide (TiO2) and immobilized metal affinity chromatography (IMAC) matrices.
- To determine the optimal combination of enrichment methods for comprehensive phosphoproteome analysis in human melanoma cells.
- To establish a robust workflow for identifying key players in the phosphoproteome cascade of A375 cells treated with Vemurafenib.
Main Methods:
- Application of four different phospho-enrichment protocols to starved A375 human melanoma cell lysates stimulated with fetal bovine serum (FBS).
- Evaluation of protocol efficiency based on peptide concentration, sulfur and phosphorus content, and liquid chromatography-mass spectrometry (LC-MS) analysis of eluted fractions.
- Comparative analysis of the phosphoproteome captured by each individual method and combinations thereof.
Main Results:
- No single phospho-enrichment method was sufficient for a comprehensive analysis of the entire phosphoproteome.
- Each tested methodology identified only a subset of the total phosphosites present in the cell lysate.
- Combining specific, efficient protocols significantly enhanced the coverage of the phosphoproteome.
Conclusions:
- A comprehensive phosphoproteome analysis requires a strategic combination of multiple enrichment methods.
- The developed workflow, utilizing optimized phospho-enrichment combinations, is effective for identifying critical components in cellular signaling pathways.
- This approach aids in pinpointing key regulators within the phosphoproteome cascade of Vemurafenib-treated A375 human melanoma cells.
