An efficient quantitation strategy for hydroxyl radical-mediated protein footprinting using Proteome Discoverer

Hydroxyl radical protein footprinting coupled with mass spectrometry has become an invaluable technique for protein structural characterization. In this method, hydroxyl radicals react with solvent exposed amino acid side chains producing stable, covalently attached labels. Although this technique yields beneficial information, the extensive list of known oxidation products produced make the identification and quantitation process considerably complex. Currently, the methods available for analysis either involve manual analysis steps, or limit the amount of searchable modifications or the size of sequence database. This creates a bottleneck which can result in a long and arduous analysis process, which is further compounded in a complex sample. Here, we report the use of a new footprinting analysis method for both peptide and residue-level analysis, demonstrated on the GCaMP2 synthetic construct in calcium free and calcium bound states. This method utilizes a customized multi-search node workflow developed for an on-market search platform in conjunction with a quantitation platform developed using a free Excel add-in. Moreover, the method expedites the analysis process, requiring only two post-search hours to complete quantitation, regardless of the size of the experiment or the sample complexity.