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

Authors

Mona Sharar, Heather Pascual, Chengzao Sun, Zhicai Shi, Guoyun Bai

Published in

Analytica chimica acta. Volume 1416. Pages 345823. Sep 22, 2026. Epub Jun 09, 2026.

Abstract

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.

PMID:
42401478
Bibliographic data and abstract were imported from PubMed on 05 Jul 2026.

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