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High Recovery Stop-and-Go Extraction Tips Using Polytetrafluoroethylene Disks Embedded with

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Summary
This summary is machine-generated.

The original StageTip disk is unavailable. Alternative StageTips using poly(styrene-divinylbenzene) (SDB) and polytetrafluoroethylene (PTFE) were tested. One alternative improved peptide identification in low-input proteomics samples.

Keywords:
StageTipbottom-up proteomicsdesaltingpeptide purificationsample pretreatment

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Area of Science:

  • Proteomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • The StageTip (stop-and-go extraction tip) is crucial for peptide purification in bottom-up proteomics.
  • The original 3M Empore disk for StageTips is no longer commercially available.
  • This necessitates evaluating alternative materials for StageTip fabrication.

Purpose of the Study:

  • To assess the performance of StageTips fabricated with alternative poly(styrene-divinylbenzene) (SDB)-containing polytetrafluoroethylene (PTFE) disks.
  • To compare the peptide recovery and identification efficiency of these alternative StageTips using varying amounts of HeLa tryptic peptides.
  • To correlate material properties of the alternative disks with their performance in trace-level proteomics.

Main Methods:

  • Fabrication of StageTips using two types of commercially available SDB-containing PTFE disks.
  • Peptide desalting and purification of 500 ng and 20 ng of HeLa tryptic digests using the fabricated StageTips.
  • Analysis of purified peptides using nanoscale liquid chromatography-tandem mass spectrometry (nLC-MS/MS).
  • Physical characterization and scanning electron microscopy (SEM) imaging of the disk materials.

Main Results:

  • Both alternative StageTips performed comparably for 500 ng peptide samples.
  • One type of StageTip identified 1.8 times more peptides from the 20 ng sample, particularly longer and more hydrophobic peptides.
  • SEM imaging revealed the high-performing disk had more PTFE fibers, partially covering SDB particles, potentially reducing hydrophobic peptide trapping.

Conclusions:

  • Disk material properties, specifically PTFE fiber content and SDB particle coverage, significantly impact StageTip performance.
  • Alternative StageTips can be effective for peptide purification, especially in low-input sample scenarios.
  • Optimizing disk material composition is key to improving peptide recovery and identification in trace proteomics.