Peptidomics has broad applications in neuroscience, including disease diagnosis, biomarker discovery, pathological mechanism investigation, therapeutic target identification, and drug development. By efficiently analyzing peptides in cerebrospinal fluid, peptidomics can uncover the underlying molecular mechanisms of central nervous system diseases, such as Alzheimer's disease and brain injuries, and identify therapeutic peptides with high specificity and low toxicity. This technology provides critical support for the diagnosis and treatment of neurological disorders, driving the development of innovative therapeutic strategies.
Case.Peptidome analysis of cerebrospinal fluid in neonates[1]
Research Background:
Hypoxic-ischemic brain injury (HIBD) is a leading cause of neonatal mortality and long-term neurological damage, with limited effective treatment options currently available. Peptides, known for their high efficacy and safety as therapeutic agents, have shown potential in various diseases but remain underexplored in the context of HIBD. Peptidomics, an emerging proteomics technology, enables efficient identification and quantitative analysis of peptides in cells, tissues, or body fluids through liquid chromatography-tandem mass spectrometry (LC-MS/MS), which is particularly suitable for detecting low-abundance and diverse protein types. In central nervous system diseases, cerebrospinal fluid (CSF) is considered a critical source for diagnostic biomarkers and therapeutic targets. Previous studies have identified numerous peptides in CSF, some of which are associated with neurodegenerative diseases. However, peptidomic analysis of CSF in neonatal HIBD remains unexplored. This identification could provide insights for the diagnosis and treatment of neonatal HIBD, laying the foundation for the development of new therapies.
Experimental Results:
A total of 35 differentially expressed peptides were identified from 25 precursor proteins. In comparison to the control group, the HIBD group exhibited upregulation of one peptide and downregulation of 34 peptides. The authors then investigated potential functional changes in CSF in HIBD by analyzing the cleavage sites of differentially expressed peptides. These cleavage sites displayed regular patterns, allowing for the analysis of proteolytic enzyme functions (as shown in Figure 1). Arginine (R) and lysine (K) were the most common cleavage sites at the N-terminal and C-terminal amino acids of precursor peptides and identified peptides, respectively. Glycine (G) was the most common cleavage site at the C-terminal amino acid of precursor peptides. Lysine (K) and isoleucine (I) were the most common cleavage sites at the N-terminal amino acid of identified peptides.
Figure 1: Cleavage Sites in Differentially Expressed Peptides
In order to preliminarily explore the potential functions of these differentially expressed peptides in neonatal HIBD, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted based on precursor proteins. The cellular components of peptide precursors were primarily associated with nuclear chromatin, nucleosome, and chromosomal parts. The most relevant biological processes included chromatin remodeling, chromatin modification, and ATP-dependent chromatin remodeling. The molecular functions of peptide precursors mainly involved chromatin binding, chromatin DNA binding, structure-specific DNA binding, and DNA binding. Pathway analysis revealed that the most relevant pathways associated with these peptide precursors were hypertrophic obstructive cardiomyopathy (HCM), leukocyte transendothelial migration, gastric acid secretion, and arrhythmogenic right ventricular cardiomyopathy (ARVC).
Figure 2: GO and KEGG Analysis of Peptide Precursors
In order to identify key peptides playing a crucial role in HIBD, the functions of differentially expressed peptides and their precursor proteins were investigated using the STRING database and UniProt database. The protein-protein interaction (PPI) network of precursor proteins is shown in Figure 3. The interactions among these precursor proteins were studied, revealing that heat shock protein 90-alpha (HSP90α/HSP90AA1) occupies a central position in the network and exhibits intricate relationships with other proteins. One of the downregulated peptides in neonatal HIBD is the 2671.5 Da peptide (HSQFIGYPITLFVEKER), which is a fragment of heat shock protein 90-alpha (HSP90α/HSP90AA1). It has been named Hypoxic-Ischemic Brain Damage-Associated Peptide (HIBDAP). Through cellular experiments, the authors speculate that HIBDAP may counteract protective factors in neonatal HIBD through cellular apoptotic mechanisms.
Figure 3: Protein-Protein Interaction Network of Peptide Precursors
This study utilized LC–MS/MS to identify differentially expressed peptides between neonatal HIBD and control group CSF samples, resulting in the identification of 35 differentially expressed peptides. This is the first study to demonstrate differentially expressed peptides in CSF between neonatal HIBD and controls. Furthermore, several significant peptides, including HIBDAP, were discovered, suggesting potential key roles in neonatal HIBD. The molecular mechanisms and biological functions of these peptides may offer new insights into the pathogenesis and therapeutic targets for neonatal HIBD.
Reference
- Hou, Xuewen et al. "Peptidome analysis of cerebrospinal fluid in neonates with hypoxic-ischemic brain damage." Molecular brain vol. 13,1 133. 2 Oct. 2020.
