Label-free quantification of cumulative cytosol-enriched peptide concentrations by mass spectrometry

The development of label-free cell permeability assays for the detection and quantification of peptide therapeutics remains analytically challenging. Accurate and rapid evaluation of target-agnostic peptides and other beyond-rule-of-five (BRO5) compounds in the pharmaceutical industry would accelerate discovery programs aimed at intracellular targets by enabling earlier prioritization based on intracellular availability. We report a label-free assay designed to quantify intact intracellular peptides, rank-order permeability, and assess intracellular stability. Cumulative peptide levels were quantified from a cytosol-enriched soluble intracellular fraction for azide-modified cell-penetrating peptides and for designed macrocycles; an impermeable negative control was included to benchmark assay specificity. Multiple cell lines were evaluated, and internalized peptides were measured over time using electrospray mass spectrometry (ESI-MS). Total ion count (TIC) for cell lysate served as a quantitative normalization tool; it mitigated the discrepancies that can originate from cell count and cell lysis efficiency. MS permeability rankings correlated 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 (P_app) values for the macrocycles, supporting external validity. This label-free assay discriminates permeable from impermeable peptides across two cell lines, quantifies intact internalized peptides without routine use of isotopically labeled internal standards. The end-to-end protocol requires ∼ two days from cell seeding to data acquisition, making it suitable for medium-throughput comparative studies in peptide lead triage.