Technical phosphoproteomic and bioinformatic tools useful in cancer research

Phosphoproteomics plays an important role in our understanding of how phosphorylation participates in translating distinct signals into the normal and or abnormal physiological responses, and has shifted research towards screening for potential therapies for diseases and in-depth analysis of phosphoproteomes. These issues can also be studied by structural analysis of proteins and bioinformatic tools. Specific domains discriminate between the phosphorylated vs. the non-phosphorylated state of proteins, based on the conformational changes induced by the presence of a negatively-charged phosphate group in the basal state of the phosphopeptide [1]Phosphorylated proteins, chemically quite stable, are prone to enzymatic modification, so that when tissues or cells are lysed, it is very likely that further enzymatic reactions will occur [2]. Good sample preparation is the key to successful analysis. These will generally be snap-frozen and treated with phosphatase inhibitors to avoid modifying phosphopeptides during sample work-up [3,4]. Also, it is critical to avoid salts and detergents, which can decrease the recovery of phosphopeptides or interfere with subsequent analysis [5]. Phosphopeptides generally make up a small portion of the peptides in a given protein sample, making detection difficult. Their enrichment [e.g. via Immobilised metal ion affinity chromatography (IMAC), Titanium dioxide metal-based chromatography (TiO2), Zirconium dioxide (ZrO2), Sequential elution from IMAC (SIMAC) or Calcium phosphate precipitation] helps to combat this problem.When combining the previously mentioned phosphoenrichments with Strong cation and anion exchange (SCX and SAX) or Hydrophilic interaction chromatography (HILIC), large-scale phosphoproteomic studies of interest can be carried out successfully [6]. If the goal of the research study includes quantification of phosphorylated proteins, there are several useful techniques [e.g. Stable Isotope Labelling with Amino acids in cell C