Introduction: For the PTM analysis using MS, protein is usually digested into peptides with proteases. The detection of these peptides is largely depending on their size; larger peptides tend to have low ionization efficiency, whereas smaller peptides can not be well separated by LC. Therefore, the peptide size suitable for detection need to be controlled by choosing appropriate protease to identify the PTM structure of the proteins. In our previous study, the immobilized protease technique was shown to be useful for the protein digestion. Here, we applied this technique to the multiple protease digestion and evaluated its effectiveness in enhancing detection efficiencies of PTM's.
Methods: Three kinds of proteases (trypsin, LysC, V8) were immobilized on the single solid surface, and used to digest BSA, beta casein and ovalbumin as model proteins. To accelerate proteolysis reaction by sample convection, the vibration reaction unit, which was developed in our previous study (Anal. Chem. Insights, 2007, 2, 69-74), was used. Digested proteins were analyzed using LC-MS. The number of the identified peptides, protein score, sequence coverage, and identification of the phosphopeptides were compared.
Results: Digestion using different proteases resulted in the identification of different peptide sequences. Consequently, the combination of these individual results provided higher sequence coverage, suggesting that the protein digestion using multiple proteases is effective to improve the accuracy of protein identification in the MS analysis. The detection of the phosphopeptides changed depending on the proteases used for the analysis; phosphopeptides from beta-casein and ovalbumin were identified only in their LysC and V8 digests, respectively. Therefore, it suggests that the utilization of the multiple kinds of proteases should be considered for the protein phosphorylation analysis.
