To investigate whether phospholipids are retained after passing through the phospholipid trap column, we measured lipids on both the standard two-valve and the extended three-valve HDX-MS system. Initially, we injected four 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) aggregates (0.01, 0.1, 1, and 10 pmol) in duplicates and plotted the obtained MS signal against the amount of lipid (Figure ). After that, we increased the concentration of POPC and ran triplicates over the delipidation system (three-valve configuration), applying both the ZrO 2 and the TiO 2 column. The remaining POPC signal after phospholipid trapping was used to calculate the delipidation efficiencies for the applied column/system. The extracted ion chromatogram (EIC) as well as the MS signal of POPC impressively illustrates a >100-1,000-fold lipid reduction for both bead types, with ZrO 2 outperforming TiO 2 (Figure ). We performed similar lipid trapping experiments applying an Escherichia coli lipid extract (EPL) and Fos-choline-12. Again, we plotted the intensity of the lipid signal against the injected amount (Figure ) and exploited the remaining MS signal to calculate the system's delipidation capacity (Table 1, S6, S7; Figure ). For EPL, we determined lipid removal efficiency for each lipid individually, which is ~96% for ZrO 2 and ~87% for TiO 2 , slightly lower than for POPC (Table , S6, S7). Both bead types, however, do not show any discrimination in terms of lipid length (Figure ). In standard HDX-MS analysis of membrane proteins, detergents are usually added to the quench buffer to facilitate nanodisc rupture . Hence, we also determined the delipidation capacity in presence of detergents, e.g. 0.1% DDM or 0.1% Fos-choline-12 (a concentration above their critical micelle concentration). DDM causes a significant drop of the delipidation efficiency (32% and 60% of POPC removal for ZrO 2 and TiO 2 respectively, Table ), potentially due to steric hindrance through a bigger micelle formation. However, Fos-choline-12 exerts no detrimental effect in terms of measured lipid removal. Moreover, despite structural similarities with phospholipids, i.e. the phosphatidylcholine headgroup, TiO 2 fails to retain Fos-choline-12, while ZrO 2 shows an even stronger binding than for POPC (Table ; Figure ). Therefore, while the presence of DDM in the quench buffer appears disadvantageous, Fos-choline-12 appears highly suitable as delipidationcompatible quench buffer additive, as ZrO 2 beads also prevent the disadvantageous Fos-choline-12 contamination of the downstream chromatography and MS source. Current workflows perform the delipidation step offline from the UPLC system , which greatly differs to the online chromatographic approach presented here. Comparing both approaches in terms of their lipid removal capacity reveals a better performance of the column-based workflow introduced here (Table ), adding another advantage to the automation benefit.