Influence of Bead Chemistry and Protein-to-Bead Ratio on the Efficiency of Solid-Phase Proteolysis

Single-pot, solid-phase-enhanced sample preparation (SP3) is a rapid, automatable, and cost-effective protein cleanup technique facilitated by the aggregation and digestion of proteins on paramagnetic beads. In this study, we document how plasma protein-to-bead ratio influences the performance in terms of the number of identified peptides and proteins for two chemically modified paramagnetic SP3 beads, i.e., Carboxylate-Speedbeads and MagReSyn-Hydroxyl beads. The optimized protein-to-bead ratio enabled the identification of 17% more plasma protein groups than the nonoptimized condition. Furthermore, we evidenced differences in the quantification results of peptides upon aggregation on hydroxyl- and carboxylate-modified beads. By assessing the physicochemical properties of these peptides, significant differences were revealed in their pI values, charge, polarity, and aspartic and glutamic acid composition. Our results highlight that the choice of beads and protein-to-bead ratio are two important parameters that require optimization depending on the physicochemical properties of the targeted proteins.