Columns for chromatographic phospholipid trap column (ZrO 2 or TiO 2 beads) and protein digestion (Pepsin agarose resin, Cat No. 20343; Thermo Fisher) were packed in-house using both a Microbore Guard Column (1.0 mm ID x 2 cm unpacked; Part No. C-128) and an Analytical Guard Column (2.0 mm ID x 2 cm unpacked; Part No. C-130B) from UVISION Technologies (London, UK). Beads were resuspended and washed in solvent A (0.23% formic acid in H 2 O, pH 2.5). Column parts were cleaned by sonication in solvent A. The column was assembled without the frit (Figure ) on the side from which the column was packed using a syringe with an appropriate adapter. After packing, the missing frit was inserted, and the column was flushed back-to-back with solvent A by applying a constant pressure with the ASM for a couple of minutes, allowing the bead matrix to settle.