Gel-BasedSampleFractionation with SP3-Purificationfor Top-Down Proteomics

Precise prefractionation of proteome samples is a potent method for realizing in-depth analysis in top-down proteomics. PEPPI-MS (Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS), a gel-based sample fractionation method, enables high-resolution proteome fractionation based on molecular weight by highly efficient extraction of proteins from polyacrylamide gels after SDS-PAGE separation. Thereafter it is essential to effectively remove contaminants such as CBB and SDS from the PEPPI fraction prior to mass spectrometry. In this study, we developed a complete, robust, and simple sample preparation workflow named PEPPI-SP3 for top-down proteomics by combining PEPPI-MS with the magnetic bead-based protein purification approach used in SP3 (single-pot, solid-phase-enhanced sample preparation), now one of the standard sample preparation methods in bottom-up proteomics. In PEPPI-SP3, proteins extracted from the gel are collected on the surface of SP3 beads, washed with organic solvents, and recovered intact with 100 mM ammonium bicarbonate containing 0.05% (w/v) SDS. The recovered proteins are subjected to mass spectrometry after additional purification using an anion-exchange StageTip. Performance validation using human cell lysates showed a significant improvement in low-molecular-weight protein recovery with a lower coefficient of variation compared to conventional PEPPI workflows using organic solvent precipitation or ultrafiltration.