Biology subjects

Rozans, S. J.

Publications and source records attributed to Rozans, S. J..

3 recordsLinked to original sources

bioRxiv2024

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.

bioengineering↗

bioRxiv2024

Optimizing Peptide Crosslinks for Cell-Responsive Hydrogels

Cells dynamically modify their local extracellular matrix by expressing proteases that degrade matrix proteins. This enables cells to spread and migrate within tissues, and this process is often mimicked in hydrogels through the incorporation of peptide crosslinks that can be degraded by cell-secreted proteases. However, the cleavage of hydrogel crosslinks will also reduce the local matrix mechanical properties, and most crosslinking peptides, such as the widely used GPQGIWGQ "PanMMP" sequence, lead to bulk degradation of the hydrogel. A subset of proteases are localized to the cell membrane and are only active in the pericellular region in the immediate vicinity of the cell surface. These membrane-type proteases have important physiological roles and enable cells to migrate within tissues. In this work we developed an approach to identify and optimize peptide sequences that are specifically degraded by membrane-type proteases. We utilized a proteomic screen to identify peptide targets, and coupled this with a functional assay that both quantifies peptide degradation by individual cell types and can elucidate whether the peptides are primarily cleaved by soluble proteases or membrane-type proteases. We then used a split-and-pool synthesis approach to generate more than 300 variants of the target peptide to improve the degradation behavior. We identified an optimized peptide sequence, KLVADLMASAE, which is primarily degraded by membrane-type proteases, but enables both endothelial cells and stem cells grown in KLVADLMASAE-crosslinked hydrogels to spread and have viabilities similar to the gels crosslinked by the PanMMP peptide. Notably, the biological performance of the KLVADLMASAE peptide-cross linked gels was significantly improved from the initial peptide target found in the proteomic screen. This work introduces a functional approach to identifying and refining protease-substrate peptides as a way to enhance the properties of hydrogel matrices.

bioengineering↗

bioRxiv2024

Quantifying and controlling the proteolytic degradation of cell adhesion peptides

Peptides are widely used within biomaterials to improve cell adhesion, incorporate bioactive ligands, and enable cell-mediated degradation of the matrix. While many of the peptides incorporated into biomaterials are intended to be present throughout the life of the material, their stability is not typically quantified during culture. In this work we designed a series of peptide libraries containing four different N-terminal peptide functionalizations and three C-terminal functionalization to better understand how simple modifications can be used to reduce non-specific degradation of peptides. We tested these libraries with three cell types commonly used in biomaterials research, including mesenchymal stem/stromal cells (hMSCs), endothelial cells, and macrophages, and quantified how these cell types non-specifically degraded peptide as a function of terminal amino acid and chemistry. We found that peptides in solution which contained N-terminal amines were almost entirely degraded by 48 hours, irrespective of the terminal amino acid, and that degradation occurred even at high peptide concentrations. Peptides with C-terminal carboxylic acids also had significant degradation when cultured with cells. We found that simple modifications to the termini could significantly reduce or completely abolish non-specific degradation when soluble peptides were added to cells cultured on tissue culture plastic or within hydrogel matrices, and that functionalizations which mimicked peptide conjugations to hydrogel matrices significantly slowed non-specific degradation. We also found that there were minimal differences across cell donors, and that sequences mimicking different peptides commonly-used to functionalized biomaterials all had significant non-specific degradation. Finally, we saw that there was a positive trend between RGD stability and hMSC spreading within hydrogels, indicating that improving the stability of peptides within biomaterial matrices may improve the performance of engineered matrices.

bioengineering↗