Phosphorylation Site Mapping Service
Single Phosphoprotein Analysis from 1D Gel Bands
The 1D band should have good intensity by colloidal coomassie staining in order to proceed. This gives us a better chance of identifying the sites of phosphorylation as the level of protein usually gives no indication of the stoichiometry of the phosphorylation present. First step in the sample submission process is usually to email the facility with the full protein sequence including any tags or linkers along with a gel image of the gel band in order to determine how well the protein will digest with trypsin and the amount of protein available.
In regards to the sample preparation prior to the gel processing please include phosphatase inhibitors as soon as you can to maintain the phosphorylation status of your protein of interest.
Gel bands are processed using our optimised in-gel digestion protocol that includes in-gel reduction/alkylation, in-gel trypsin digestion followed by peptide extraction. Extracted peptides are then run by nLC-MS/MS e.g. Q-Exactive Plus. Analysis of the MS/MS data identify the protein and sites of phosphorylation by database searching using Proteome Discoverer (Thermo Scientific) and Mascot (Matrix Science). Results of protein identification and sites of phosphorylation are presented in the form of an annotated excel spreadsheet.