Gel-Based Sample Fractionation with SP3-Purification for 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.