A New High-Throughput Method to Measure How Well Peptides Get Inside Cells | Research

The takeaway

Researchers developed a rapid, label-free assay called CAMPA that uses mass spectrometry and hydrogen-deuterium exchange to rank how easily peptides cross cell membranes, successfully testing 24 diverse peptides.

24 peptides ranked
Including cell-penetrating, stapled, and macrocyclic peptides — all validated against published permeability data

What the researchers found

The Cell-based Approach Membrane Permeability Assay (CAMPA) successfully ranked the cell membrane permeability of 24 diverse peptides — including cell-penetrating peptides, stapled peptides, and macrocyclic peptides — using live THP-1 and AsPc-1 cells.

The method works by briefly exposing peptide-cell mixtures to deuterium oxide. Peptides outside cells undergo hydrogen-deuterium exchange and gain mass, while peptides inside cells are shielded from labeling. MALDI mass spectrometry detects the ratio of labeled to unlabeled peptides over time. Results correlated with previously published permeability data. The assay also distinguished between passive diffusion and active (endocytosis-mediated) transport when an endocytosis inhibitor was added.

Why it matters

Peptide drugs are increasingly being developed to hit intracellular targets like transcription factors and protein-protein interactions, but most peptides struggle to cross cell membranes. Having a fast, reliable way to measure cell permeability early in drug development can help scientists prioritize the most promising candidates and avoid wasting resources on peptides that cannot reach their targets inside cells.

How the study worked

Peptides were incubated with live THP-1 (human monocytic) and AsPc-1 (pancreatic cancer) cells. At set time points, samples were briefly exposed to deuterium oxide to label extracellular peptides via hydrogen-deuterium exchange. Intracellular peptides were protected from labeling. MALDI mass spectrometry measured the ratio of deuterium-labeled (extracellular) to unlabeled (intracellular) peptides. The entire workflow was automated, including data processing via custom Python scripts. To differentiate passive from active transport, selected experiments included endocytosis inhibitors.

What this study cannot tell us

The assay was tested with only 24 peptides across two cell lines, which may not capture the full diversity of peptide chemistries or cell types relevant to drug development. The method relies on short deuterium exposure times that may not perfectly quantify permeability for very fast or very slow-permeating peptides. MALDI-MS requires specialized equipment not available in all labs. The approach measures total cell entry but does not reveal where inside the cell peptides accumulate (e.g., cytoplasm vs. endosomes).

How to read the evidence

This is a methodological study demonstrating a new analytical technique. It is not a therapeutic study, so traditional evidence grading (clinical trial levels) does not directly apply. The method was validated against literature data for 24 peptides across two cell lines, providing reasonable proof of concept.

When this study was published

Published in 2022, this method addresses an ongoing need in peptide drug development. As the peptide therapeutics field continues to grow, tools like CAMPA remain highly relevant.

The bigger picture

The pharmaceutical industry is investing heavily in peptide and macrocyclic therapeutics that can target previously 'undruggable' intracellular proteins. A major bottleneck has been the lack of fast, reliable permeability assays — existing methods often require fluorescent labels that can alter peptide behavior, or are too slow for high-throughput screening. CAMPA addresses this gap by providing a label-free, automated, and relatively rapid method that could accelerate the peptide drug discovery pipeline.

Questions still open

Can CAMPA be adapted to measure peptide permeability across tissue barriers like the intestinal epithelium or blood-brain barrier?
How does the method perform with larger or more hydrophobic peptides that might interact with the cell membrane without fully crossing it?
Could CAMPA be integrated into automated drug screening platforms to test hundreds or thousands of peptide candidates simultaneously?

Common questions

Why is it so hard to measure whether peptides can get inside cells?

Most existing methods require attaching a fluorescent label to the peptide, which can change its size, charge, and behavior — potentially altering the very permeability you're trying to measure. Other methods are slow or require large amounts of material. CAMPA solves this by using the peptide's own mass as the readout, avoiding the need for any chemical modification.

What types of peptides were tested with this method?

The researchers tested 24 diverse peptides including cell-penetrating peptides (naturally good at crossing membranes), stapled peptides (chemically locked into helical shapes), and macrocyclic peptides (ring-shaped molecules). This diversity helped validate that the method works across different peptide architectures, not just one type.

Read the original research

Rapid label-free cell-based Approach Membrane Permeability Assay using MALDI-hydrogen-deuterium exchange mass spectrometry for peptides.

Analytica chimica acta, 1225, 340234

View on PubMed ↗Publisher’s paper ↗

Citation

Makarov, Alexey A; Jiang, Yuan; Sondey, Christopher; Zhang, Minjia; Mansueto, My Sam; Pirrone, Gregory F; Huang, Chunhui; Biswas, Kaustav; Duggal, Ruchia; Al-Sayah, Mohammad Ahmed; Regalado, Erik L; Mangion, Ian. (2022). Rapid label-free cell-based Approach Membrane Permeability Assay using MALDI-hydrogen-deuterium exchange mass spectrometry for peptides.. Analytica chimica acta, 1225, 340234. https://doi.org/10.1016/j.aca.2022.340234