Tobias Hedtke (Halle a. d. Saale, DE), Dr. Andrea Friedmann (Halle a. d. Saale, DE), Christian Ihling (Halle a. d. Saale, DE), Dr. Christian Schmelzer (Halle a. d. Saale, DE)
Abstract text (incl. figure legends and references)
Analyzing bioactive peptides released from collagen-based biomaterials is of great significance in biomedical applications. The release of bioactive peptides from these biomaterials can modulate various biological processes and can stimulate tissue regeneration. Therefore, understanding the composition and activity of these peptides released by the degradation of collagen-based scaffolds is crucial for designing and optimizing these materials for specific biomedical applications.
In this proof-of-concept study, Tandem Mass Tag labeling was used to detect differences in the composition of two batches of collagen peptides that differed in bioactivity. Collagen peptides generated by alcalase treatment were dissolved in 50 mM HEPES buffer, labeled with TMT Duplex reagent (Thermo Fisher) and pooled. A TMT-labeled tryptic BSA digest was used for validation. All samples were analyzed by liquid chromatography-coupled mass spectrometry and subsequent peptide sequencing.
The validation of the method with trypsinized BSA showed that 95.2% of all peptides were within a 20% deviation window around the expected value. Analogous, 92.2% and 85.6% of all peptides were within a 15% and 10% deviation window, respectively. Tripeptides were the smallest peptides detected in this study. Differences in the peptide release pattern were detectable primarily in regard of the hydrophobicity of the peptide sequences between the two alcalase digests. It is reasoned that the relative number of hydrophobic peptides is causal for an increased bioactivity.
It has been proven that the relative analysis of peptides released from protein-based materials can be done using TMT labels with minimal effort in sample preparation. This versatile tool is capable of detecting differences in the pattern of peptide release between samples of different biological activity and is capable of elucidating the mode of action through the analysis of the peptide sequences.