Question

Cold acclimation is able to enhance the cold tolerance of a crop. Cold temperature induces phosphoproteome alteration, and the cold-induced protein phosphorylation associates with the cold tolerance. Describe a workflow (or steps) of 4C quantitative PTM proteomics used to identify phosphosites that associate with cold tolerance of a banana plant. These phosphosites might be used as biomarkers when people are trading banana seedlings on markets.

Answer 1

• 2D-PP mapping is one of the most common type of both quantitative and qualitative phosphorylation.
• In this method, the peptides are separated by electrophoresis on a thin layer cellulose plate and then by thin-layer chromatography (TLC) technique on the same plate.
• Separated 32P radiolabeled phosphopeptides are detected using autoradiography and phospho imaging.
• The number of spots seen in autoradiography is an estimate of the maximum number of phosphorylated sites.
• The spot intensity provides an indication of the relative stoichiometry of phosphorylation between the peptides.
• An advantage of is that this method produces purified phosphopeptides that can be analyzed after their extraction from the plate directly by mass spectrometry-based methods.