High-Density Immobilization of TCEP on Silica Beads for Efficient Disulfide Reduction and Thiol Alkylation in Peptides

Tris-(2-carboxyethyl)phosphine (TCEP) linked to agarose beads is widely used for reducing disulfide bridges in proteins and peptides. The immobilization of TCEP on beads allows efficient removal after reduction to prevent its reaction with alkylating reagents and thus interference with conjugation reactions. However, a limitation of agarose TCEP is its relatively low reduction capacity per milliliter of wet beads (about 15 mu mol/ml), making it unsuitable for the reduction of disulfides from molecules at millimolar concentrations. In this work, we tested the immobilization of TCEP to a range of different solid supports and found that conjugation to silica gel offers TCEP beads with about 8-fold higher reduction capacity (129 +/- 16 mu mol/ml wet beads). We show that it allows reducing disulfide-cyclized peptides at millimolar concentrations for subsequent cyclization by bis-electrophile linker reagents. Given the substantially higher reduction capacity, the robust performance in different solvents, the low cost of the silica gel, and the ease of functionalization with TCEP, the silica gel-TCEP is suited for reducing disulfide bridges in essentially any peptide and is particularly useful for reducing peptides at higher concentrations.|TCEP immobilized on agarose is widely used for efficient reduction of cysteines in peptides and proteins, but the reducing capacity is rather low. Herein, we have compared different solid supports for TCEP immobilization and found that silica-TCEP has an 8-fold higher reduction capacity, allowing thiol reduction of peptides at millimolar concentrations.image