High Recovery Stop-and-Go Extraction Tips Using Polytetrafluoroethylene Disks Embedded with Poly(styrene-divinylbenzene) Particles for Proteomics

ABSTRACT The stop-and-go extraction tip (StageTip) is widely used for peptide purification in bottom-up proteomics, yet the original 3M Empore TM disk is no longer available, prompting the need to evaluate current alternatives. Two types of commercially available poly(styrene-divinylbenzene) (SDB)-containing polytetrafluoroethylene (PTFE) disks were used to fabricate StageTips. Desalting was performed on 500 ng and 20 ng of HeLa tryptic peptides, and the nanoLC/MS/MS results were compared. Both StageTips demonstrated equivalent performance for the 500 ng sample, but for the 20 ng sample, one StageTip identified 1.8 times more peptides, particularly longer and more hydrophobic peptides. Physical characterization and scanning electron microscope imaging of these disks revealed that the high-performing disk contains more PTFE fibers, partially covering the SDB particle surface. This likely prevents hydrophobic peptides from being trapped in small mesopores, improving recovery from low-input samples. These findings demonstrate that disk material properties critically influence performance in trace proteomics.