HYPer-SPM: Ultra Efficient Peptide Purification by a Pipette-tip Microcolumn Enabled by Hydrophobic Microparticles Hybridyzed Sponge-like Monolithic Carrier

Peptide purification techniques with minimal loss play a crucial role in enhancing sensitivity and coverage in mass spectrometry (MS)-based proteomics. Here, we developed a pipette tip-based microcolumn with hydrophobic particles embedded in a sponge-like monolithic carrier, named HYPer-SPM, achieving highly efficient peptide recovery in peptide purification by solid-phase extraction (SPE). HYPer-SPM was synthesized by thermal mixing thermoplastic resins of ethylene-vinyl acetate copolymer (EVA) with styrene-divinylbenzene copolymer (St-DVB) particles. The column packing process of HYPer-SPM was easy and reproducible due to its sponge-like flexibility, serving simultaneously as a column material and frit in a pipette tip. Peptide samples were comprehensively retained on the hydrophobic surface of HYPer-SPM, while thermally melted EVA resins effectively prevented the irreversible adsorption of peptides into the mesopores of St-DVB particles by filling them. The unique morphology of HYPer-SPM resulted in the identification of more than double the number of peptides compared to the standard StageTip method. Furthermore, the commonly identified peptide intensities were consistently higher in HYPer-SPM over the entire retention time range. The highly efficient peptide recovery of HYPer-SPM was especially emphasized for hydrophobic and longer peptides. Our HYPer-SPM holds great promise as a peptide purification method in ultra-sensitive proteomics, finding potential applications in clinical proteomics, single-cell proteomics, and deep visual proteomics by integrating other sample preparation methods.