BAC-DROP: Rapid Digestion of Proteome Fractionated via Dissolvable Polyacrylamide Gel Electrophoresis and Its Application to Bottom-Up Proteomics Workflow
Ayako Takemori, Jun Ishizaki, Kenji Nakashima, Takeshi Shibata, Hidemasa Kato, Yoshio Kodera, Tetsuro Suzuki, Hitoshi Hasegawa, Nobuaki Takemori
Abstract
'-bis(acryloyl)cystamine (BAC) cross-linked gels that can be solubilized by reductive treatment. Making use of an established workflow called BAC-DROP (BAC-gel dissolution to digest PAGE-resolved objective proteins), crude proteome samples were fractionated based on molecular weight by BAC cross-linked PAGE. After fractionation, the gel fragments were reductively dissolved in under 5 min, and in-solution trypsin digestion of the protein released from the gel was completed in less than 1 h at 70 °C, equivalent to a 90-95% reduction in time compared to conventional in-gel trypsin digestion. The introduction of the BAC-DROP workflow to the MS assays for inflammatory biomarker CRP and viral marker HBsAg allowed for serum sample preparation to be completed in as little as 5 h, demonstrating successful marker quantification from a 0.5 μL sample of human serum.