Rozans Samuel J, Moghaddam Abolfazl S, Pashuck E Thomas
Department of Bioengineering, Lehigh University, Bethlehem, PA, USA, 18015.
bioRxiv. 2024 Oct 15:2024.10.11.617883. doi: 10.1101/2024.10.11.617883.
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.
由于肽易于合成、能够向细胞发出信号以及可改变生物材料的性质，因此在生物材料中得到广泛应用。使用肽的一个关键优势在于，它们是细胞分泌酶的天然底物，这为利用细胞分泌酶来调节细胞与材料的相互作用创造了可能性。然而，这些酶也可能导致生物材料中或肽疗法中的生物活性肽发生不必要的降解。液相色谱 - 质谱联用（LC-MS）是一种广泛使用的强大方法，它能够分离复杂的分子混合物，并在一次运行中对多种分析物进行定量。在使用LC-MS对细胞诱导的肽降解进行多重定量时存在若干挑战，包括需要不可降解的内标以及确定最佳样品储存条件。另一个问题是，细胞培养基和生物样品通常同时含有蛋白质和脂质，它们会在色谱柱上积累并降低其性能。然而，去除这些成分可能成本高昂、耗时且会增加样品变异性。在此我们表明，直接将样品注入LC-MS而无需任何纯化处理能够快速准确地定量肽浓度，并且在一根色谱柱上可以进行数百次LC-MS运行，而不会显著降低对肽库降解进行定量的能力。我们还表明，当亲水性肽无法保留在色谱柱上时，色谱柱失效就很明显，并且使用标准肽混合物进行色谱柱基准测试可以很容易地识别这一点。总的来说，这项工作引入了一种简单有效的方法，用于同时定量细胞培养中数十种肽的降解。通过提供一种简化且经济高效的方法来直接定量复杂生物样品中的肽降解，这项工作能够更有效地评估肽的稳定性和功能，促进先进生物材料和基于肽的疗法的开发。
