A Streamlined High-Throughput LC-MS Assay for Quantifying Peptide Degradation in Cell Culture.

Peptides are widely used in biomaterials due to their easy of synthesis, ability to signal cells, and modify the properties of biomaterials. A key benefit of using peptides is that they are natural substrates for cell-secreted enzymes, which creates the possibility of utilizing cell-secreted enzymes for tuning cell-material interactions. However, these enzymes can also induce unwanted degradation of bioactive peptides in biomaterials, or in peptide therapies. Liquid chromatography-mass spectrometry (LC-MS) is a widely used, powerful methodology that can separate complex mixtures of molecules and quantify numerous analytes within a single run. There are several challenges in using LC-MS for the multiplexed quantification of cell-induced peptide degradation, including the need for non-degradable internal standards and the identification of optimal sample storage conditions. Another problem is that cell culture media and biological samples typically contain both proteins and lipids that can accumulate on chromatography columns and degrade their performance. However, removing these constituents can be expensive, time consuming, and increases sample variability. Here we show that directly injecting samples onto the LC-MS without any purification enables rapid and accurate quantification of peptide concentration, and that hundreds of LC-MS runs can be done on a single column without a significantly diminish the ability to quantify the degradation of peptide libraries. We also show that column failure is evident when hydrophilic peptides fail to be retained on the column, and this can be easily identified using standard peptide mixtures for column benchmarking. In total, this work introduces a simple and effective method for simultaneously quantifying the degradation of dozens of peptides in cell culture. By providing a streamlined and cost-effective method for the direct quantification of peptide degradation in complex biological samples, this work enables more efficient assessment of peptide stability and functionality, facilitating the development of advanced biomaterials and peptide-based therapies.