Finally, we determined the ideal glycine-HCl concentration in the quench buffer. The best performances, measured in terms of peptide signal intensity and identifications, are obtained with 200 and 500 mM glycine-HCl quench buffers, with no significant differences between the two concentrations. To note, the average peptide signal drops at higher concentrated glycine-HCl quench buffers, which might be explained by peptide ion suppression due to uncomplete desalting or a detrimental effect on the protein digestion (Figure ). We then compared the performance of our optimized online delipidation system, in terms of protein recovery, to the off-line ZrO 2 -based delipidation protocol proposed in the literature . Although a higher amount (25 mg) of beads is used to pack the phospholipid trap column in the online system compared to the 10 uL (3 mg) used in the off-line workflow, in our hands unspecific adsorption in the offline protocol was much higher compared to the automated workflow with glycine-HCl and/or the blockage of the beads (Figure ). This experiment highlights that this problem requires to be addressed when beads are handled manually (off-line), as reliable measurements are hardly feasible without an adequate strategy to overcome unspecific protein binding. We envision that the BSA-blockage of beads and the use of glycine-HCl as quench buffer will be suitable to prevent protein unspecific adsorption to beads also in the offline workflow.