The integration of an additional column into the delipidation system might lead to unfavorable, unspecific interactions between the target protein and the phospholipid trap column matrix. To investigate such unspecific adsorption effects, we conducted bottom-up PhosB measurements on both systems, i.e. standard two-valve and extended three-valve HDX-MS configuration. We solubilized PhosB in 10 mM potassium phosphate (pH 7.0) and diluted it in 100 mM potassium phosphate (pH 2.3), simulating HDX quench conditions (final pH of 2.5 at 0 C). Then, we compared the peptide intensities obtained before and after passing through the TiO 2 or ZrO 2 column. We measured the extent of unspecific binding of PhoB to the phospholipid trap column on both protein and peptide level, as the phospholipid trap column can be operated up-and downstream the protease column. Unfortunately, the degree of unspecific column:peptide and in particular column:protein interactions were significant (Figure ), making reliable peptide measurements difficult not to say impossible. Put simply, unspecific adsorption effects are more severe for intact protein than on the peptide level. Only TiO 2 :peptide interactions seem to be mostly negligible. On the protein level, the loss in intensity amounts to two orders of magnitude on average independent of the bead type, which decreases the signal-to-noise ratio to an unacceptable degree for most peptides. We also calculated the percentage of detected peptides relative compared to control measurements performed in the two-valve configuration. The loss of identifiable peptides ranges from 15 to 30% on the protein level for both bead types and for the ZrO 2 :peptide configuration (Figure ). Solely the setup for TiO 2 :peptide measurements is acceptable in terms of peptide loss. The addition of a chaotropic agent to the quench buffer, e.g. 3.0 M guanidinum-HCl (1.5 M final) as potential suppressor of unspecific protein/peptide adsorption leads to a marginal increase of signal. The number of undetected peptides however remains largely unimproved, accounting still for 15-20%.