An enhanced pressure (up to ～15,000 psi) sustainable BEH column with immobilized pepsin has been successfully applied in Waters HDX MS system. An systematically study of protein digestion efficiency and back exchange under enhanced pressure shows an improvements in mAb IgG2 digestion efficiency. IgG2 is believed to be resistant to digestion since four disulfide bonds locates in the hinge region. Our results indicated: Overall more overlapping peptides with shorter length were observed under HP digestion, which increased protein sequence coverage, and spatial resolution compared to NP under the same digestion conditions. Higher flow rate and longer desalting time minimized the salt contamination without causing more deuterium loss. Less chaotropic reagents was used under HP digestion and obtained a similar digestion efficiency.
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