Mass Spectrometric Analysis Of Bioactive Peptides

Neuropeptides are a class of endogenous biologically active peptides with a short amino acid sequence between 3 and 100 amino acids,which participates in various physiological activities in the organism,such as food intake,learning,memory,metabolism,etc.Different neuropeptides have different functions,even for neuropeptide isoforms resulting from the same family they may have distinct effect on certain biological process.Neuropeptides are produced from their protein precursors under the selective action of endopeptidases,and some of them undergo further posttranslational modifications(PTMs),which can profoundly influence their bioactivity.A neuropeptide precursor molecule may contain multiple neuropeptide sequences.Identification of neuropeptides is challenging due to low levels of concentration,easy degradation,and low molecular weight.It is also difficult to provide useful information for the identification of novel neuropeptides.With technological advancements,mass spectrometry-based technology has been widely used for large-scale identifications of proteins or peptides.The mass spectrometer generates data by detecting the mass-to-charge ratio of the protein or peptide,and then identifies them through the database search software.The intact molecular weights and amino acid sequences of neuropeptides can be identified using mass spectrometry on a large scale with high accuracy,and the molecules can be quantified.Based on these general features,we use biological mass spectrometry to study the endogenous neuropeptides.We reviewed and considered the distribution and content of neuropeptides in the brain of rats.First of all,we selected the database and data search software used for mass spectrometry data analysis.Secondly,the method of extracting neuropeptide from rat brain samples was optimized.The method of extracting neuropeptides by urea or acidified methanol was compared.In this paper,the neuropeptides contained in the hypothalamus,hippocampus,striatum and olfactory bulb of the rat brain were analyzed and identified,and the number and type of the neuropeptides were analyzed.Post-translational modification,corresponding precursor molecules and other characteristics were also characterized.The hypothalamus is a highly prominent region in the brain,which contains the most types of neuropeptides,but also contains many unique neuropeptides that do not exist in other brain regions.A number of expected neuropeptide families and known isoforms have been identified from the rat brain,demonstrating the reliability of this experiment.In this study,we conducted large scale identification and characterization of the rat neuropeptidome,whose differential expressions could offer critical insight into future development of the treatment of neurological diseases based on region specific peptide expression.The study of neuropeptides is essential for basic neuroscience and pharmacy studies,which facilitate a comprehensive understanding of the functions of different regions of the brain.The optimized extraction method for rat brain neuropeptide will provide helpful guide for other researchers to study neuropeptides of in rodent model systems as well as other model organisms.