title: Enzymatic protein digestion using a dissolvable polyacrylamide gel and its application to mass spectrometry-based proteomics.
abstract: 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.
authors: Proteo-Science Center, Ehime University, Shitsukawa, Toon City, Ehime, Japan. Electronic address: takemori@m.ehime-u.ac.jp.
authors: Nobuaki
authors: N
authors: Takemori
authors: Proteo-Science Center, Ehime University, Shitsukawa, Toon City, Ehime, Japan.
authors: Ayako
authors: A
authors: Takemori
authors: Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, Japan.
authors: Jun
authors: J
authors: Ishizaki
authors: Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, Japan.
authors: Hitoshi
authors: H
authors: Hasegawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D004330:Drosophila / Q000737:chemistry; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D014357:Trypsin / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; D010446:Peptide Fragments; D014357:Trypsin
keywords: Bis-acrylylcystamine; In-gel protein digestion; Protein quantitation; Shotgun proteomics