Optimized Sample Handling Minimizes Peptide Adsorption to Plastics to Enable High Sensitivity Evosep Based Chemical...

Chemical proteomics approaches often yield low peptide recovery because they aim to enrich low-abundance proteins. Poor sample handling further reduces recovery through peptide adsorption to plastic surfaces and losses due to vacuum evaporation. We systematically mapped these losses across buffers, volumes, plastics and pH, then developed an Evotip-compatible handling protocol that minimizes adsorption and removes the need for vacuum evaporation. Losses to plastic adsorption and vacuum evaporation were most apparent at low inputs (< 200 ng) and with larger volumes (> 20 µL) but were also dependent on acidification as well as the buffer and plastic used. The optimized workflow: direct acidification of peptides, frozen storage if needed, and direct loading onto Evotips, resulted in up to ∼90-fold gains versus workflows incorporating vacuum concentration steps, a common practice in proteomics sample preparation, when peptide input was limited to 10 ng. This optimized sample handling method enables high-throughput chemical proteomics by reducing manual handling steps and enables the characterization of low abundance proteins previously lost during sample preparation.