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ABSTRACT Peptides are widely used in biomaterials due to their ease 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 nondegradable 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. Removing these constituents can be expensive, time‐consuming, and increases sample variability. However, loading unpurified samples onto the column without removing lipids and proteins will foul the column. Here, we show that directly injecting complex, unpurified 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 significantly diminishing the ability to quantify the degradation of peptide libraries. To understand how repeated injections degrade column performance, a model library was injected into the LC–MS hundreds of times. It was then determined that column failure is evident when hydrophilic peptides are no longer retained on the column and that failure can be easily identified by 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.
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Microalgae Peptide-Stabilized Gold Nanoparticles as a Versatile Material for Biomedical Applications
Microalgae peptides have many medical and industrial applications due to their functional properties. However, the rapid degradation of peptides not naturally present in biological samples represents a challenge. A strategy to increase microalgae peptide stability in biological samples is to use carriers to protect the active peptide and regulate its release. This study explores the use of gold nanoparticles (AuNPs) as carriers of the Chlorella microalgae peptide (VECYGPNRPQF). The potential of these peptide biomolecules as stabilizing agents to improve the colloidal stability of AuNPs in physiological environments is also discussed. Spectroscopic (UV-VIS, DLS) and Microscopic (TEM) analyses confirmed that the employed modification method produced spherical AuNPs by an average 15 nm diameter. Successful peptide capping of AuNPs was confirmed with TEM images and FTIR spectroscopy. The stability of the microalgae peptide increased when immobilized into the AuNPs surface, as confirmed by the observed thermal shifts in DSC and high zeta-potential values in the colloidal solution. By optimizing the synthesis of AuNPs and tracking the conferred chemical properties as AuNPs were modified with the peptide via various alternative methods, the synthesis of an effective peptide-based coating system for AuNPs and drug carriers was achieved. The microalgae peptide AuNPs showed lower ecotoxicity and better viability than the regular AuNPs.
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- PAR ID:
- 10338676
- Date Published:
- Journal Name:
- Life
- Volume:
- 12
- Issue:
- 6
- ISSN:
- 2075-1729
- Page Range / eLocation ID:
- 831
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/life12060831
