Optimized approach for active peptides identification in Cerebrolysin by nanoLC-MS

Cerebrolysin (CBL) is a peptide-rich preparation made by hydrolysis and purified extraction of porcine brain. CBL contains various neuroprotective peptides, such as neurotrophic factor, nerve growth factor and ciliary neurotrophic factor, which can be used to treat neurodegenerative diseases. However, the active peptides in CBL had not been studied in depth. In this study, the following was carried out in order to investigate the active peptides in CBL. First, CBL samples were treated using organic reagents (acetonitrile and acetone) to precipitate the proteins and different solid phase extraction methods (MCX mixed-mode cartridges, C18 SPE cartridge columns and HILIC sorbent). Then the samples were analyzed using nanoLC-MS, followed by the identification of peptides using different sequence analysis software (PEAKS, pNovo and novor). Finally, bioinformatics analysis was performed to predict peptides with potential neuroprotective functions in CBL, such as anti-inflammatory and antioxidant peptides. Results showed that the number of peptides obtained by the MCX method coupled with PEAKS was the highest and the method was the most stable. Bioinformatic analysis of the detected peptides showed that two anti-inflammatory peptides (LLNLQPPPR and LSPSLRLP) and an antioxidant peptide (WPFPR) might be neuroprotective peptides in CBL. In addition, this study found that some peptides in CBL were present in myelin basic protein and tubulin beta chain. The results of this study for the detection of active peptides in CBL laid the foundation for the subsequent study of its active ingredients.