The additional column matrix initially led to a significant amount of unspecific protein and peptide binding, which led to unfavourable loss of peptide signals. Minimizing protein loss was a crucial step and required the optimization of both bead and solution conditions. Increasing the amount of protein injected to obtain a sufficiently high MS signal, a strategy commonly applied so far, is unfavorable for two main reasons; i) the increase in sample consumption of the target membrane proteins, which are generally obtained in low amounts due to a challenging expression and purification; and ii) the proportional increase in the amount of lipid components injected, at the expense of their effective removal. We could demonstrate that such protein:bead unspecific adsorption, hence protein loss, can be largely minimized by blocking unspecific binding sites utilizing a combination of blocking reagent, e.g. BSA, and a suitable quench buffer of 200-500 mM glycine-HCl, with only a minor effect on lipid removal efficiencies. However, we note that the application of ZrO 2 beads still remains a compromise between delipidation efficiency and the prevention of unspecific protein adsorption. Engineering a different type of beads or a dedicated ZrO 2 -based trap column could potentially overcome this issue in future. Furthermore, and more generally, the optimized extended LC setup, equipped with an additional pump, could be utilized to integrate other substrate traps or chromatographic/enzymatic columns into the conventional HDX-MS apparatus, to enhance the flexibility in HDX-MS analysis of complex protein samples.