Enzymatic protein digestion using a dissolvable polyacrylamide gel and its application to mass spectrometry-based proteomics

• We developed a novel in-gel digestion method using a dissolvable gel matrix.
• Complete dissolution of the gel has the advantage of reproducible peptide recovery.
• Protein samples trapped in the gel were stable at room temperature.
• Reproducible sample recovery was obtained after storage for one week.

Enzymatic protein digestion in polyacrylamide gel has been used for sample pretreatment in mass spectrometry-based proteomics due to its effectiveness in removing contaminants that interfere with sample ionization. However, the difficulty of recovering the digested peptides from the solid gel matrix has been a drawback of this method. Here we have developed a novel in-gel digestion method to enhance peptide recovery using a dissolvable, bis-acrylylcystamine (BAC)-crosslinked polyacrylamide gel. After enzymatic protein digestion in BAC gel, we completely dissolved the gel by reductive treatment with tris-(2-carboxyethyl) phosphine to release the digested peptides from the gel. Our analysis revealed that the reductive dissolution of the BAC gel enhances the peptide recovery, which has a significantly higher protein identification capability than the conventional method, using an insoluble polyacrylamide gel. In addition, protein samples trapped in dehydrated BAC gel were stable at room temperature and reproducible sample recovery was obtained after storage for one week. These results indicate that the proposed method could be an effective tool for conducting sample pretreatment for mass spectrometry-based protein analysis.