In the field of drug discovery, proteomic approach can be a powerful procedure to explore the drug effect because in most cases, target molecules of drugs are protein. Typical flow of proteome study is well established in combination with mass spectrometry analysis and database searching followed by gel separation and in-gel digestion of proteins. In this flow, improving the in-gel digestion protocol is important to have advantage over identifying lower abundant protein because it is difficult to modify mass spectrometry hardware to improve the sensitivity of the system by ourselves. We previously suggest using thin-gel separation such as 0.5mm thickness, negative staining and addition of n-octylglucoside with trypsin treatment in order to improve the recovery of the digested peptide fragments. These combinations also worked fine in 96-well plate high throughput format since n-octylglucoside helped both enhancing digestion efficiency and prevent adsorption of the digested peptides to the plate wall. In this study, we further modified our protocols using shorter separation gel such as 4cm while length of typical gels are 8-10cm. The shorter gel separation helped to reduce the gel volume per a band, and it was expected to improve digestion efficiency as well as using thinner gel for a same reason. This strategy was applied to GeLC-MS which gel lane was divided into 4 pieces with ignoring the separation pattern of the protein, and analyzed by LC-MS after in-gel digestion. The number of identified peptides from shorter gel was much superior to that of typical gel, and in-gel digestion efficiency was apparently improved. We will further discuss the possibility using other detergent instead of n-octylglucoside in order to establish better system.
