In situ polymerized monolith tips for reproducible, format-flexible proteomic sample preparation applied to biofluids

Sample preparation increasingly sets the throughput and reproducibility of mass spectrometry (MS)-based proteomics. StageTips (stop-and-go extraction tips) and variants thereof have long been common implements to purify samples, and we recently extended the concept to solid-phase extraction capture (SPEC) tips, in which the entire digestion takes place in sub-microliter volumes. We replace the hand-packed bed with a monolith photopolymerized directly inside the pipette tip from a defined recipe (pSPEC), implemented here with strong anion-exchange (SAX) chemistry. A liquid-handling robot casts 384 tunable tips in minutes, at low cost and in any format, adding negligibly to the workflow's variance. Across biofluids, pSPEC added ∼20% more identifications than in-solution digestion of plasma and reached 3,500 protein groups from a single injection of healthy urine and 4,800 from saliva at 100 samples per day, depths usually requiring depletion or fractionation. The same light-cast approach should extend to single cells, affinity capture, and population-scale studies.