Label-free mass spectrometry assay quantifies intracellular peptides, ranks permeability, and assesses stability

Developing effective intracellular-targeting peptide therapeutics is hampered by the analytical challenge of accurately quantifying their cell permeability and intracellular availability. Traditional label-based assays can be complex or lack specificity for intact peptides, while existing methods often struggle with beyond-rule-of-five (BRO5) compounds. This gap in rapid, label-free quantification of intact intracellular peptides delays early-stage drug discovery, making it difficult to prioritize promising candidates based on their ability to reach their target within the cell.

Researchers developed a label-free assay to quantify intact intracellular peptides. They measured cumulative peptide levels from a cytosol-enriched soluble intracellular fraction using electrospray mass spectrometry (ESI-MS). The assay evaluated azide-modified cell-penetrating peptides and designed macrocycles, including an impermeable negative control for benchmarking specificity. Multiple cell lines were tested, and internalized peptides were measured over time. Total ion count (TIC) from cell lysate served as a quantitative normalization tool, mitigating discrepancies from cell count and lysis efficiency.

The label-free ESI-MS assay successfully discriminated permeable from impermeable peptides across two distinct cell lines. The method accurately quantified intact internalized peptides without requiring routine use of isotopically labeled internal standards. Importantly, the MS permeability rankings demonstrated strong external validity: > The assay's permeability rankings correlated significantly with published cellular half-maximal effective concentration (EC50) values for the azide-modified peptides and with reported parallel artificial membrane permeability assay (PAMPA) apparent permeability coefficient (Papp) values for the macrocycles. The entire end-to-end protocol, from cell seeding to data acquisition, requires approximately ~two days, making it suitable for medium-throughput comparative studies in peptide lead triage.