General in-gel protein identification workflow
First, we wash the gel pieces with successive dehydration/rehydration cycles in MS-compatible solutions. In a second step, we break intra- and inter-protein S-S bridges using a reducing agent and block the free cysteines by alkylation. The reduced proteins are then digested directly in the gel with a protease that cleaves after known residues. Following digestion, we extract the resulting peptides from the gel by successive rounds of dehydration and sonication. Finally, these peptides are purified using reversed phase SPE (solid phase extraction) and injected in the mass spectrometer.
Before entering the MS, the peptides are further separated using a liquid reversed phase chromatography system that is directly connected to the mass spectrometer. This results in a better acquisition depth, since the additional separation gives the MS enough time to adequately sequence the peptides that are the sample. Once in the mass spectrometer, peptides are fragmented in smaller pieces and recorded by the instrument's high resolution detector using a workflow called Data Dependent Acquisition (DDA). This process yields a list of precursor masses with its associated list of fragment masses.
Data analysis and protein identification
To obtain the identity of the proteins that were in the sample, we load the data in a protein identification software. This software associates the recorded list of masses to a list of peptides and attributes them to one or more proteins. Of note, this process also reports any post-translational modification, chemical modification or amino acid substitution that could be recorded.
Reports of protein identification consist of a spreadsheet with a list of probable IDs, as well as a score and the number of peptides associated with every ID. We will also provide the false discovery rate (FDR) file generated by the identification software.