Identification of novel biomarkers for neonatal hypoxic-ischemic encephalopathy using iTRAQ

Abstract

Background: A prompt diagnosis of HIE remains a challenge clinically. This study aimed to identify potential biomarkers of neonatal hypoxic-ischemic encephalopathy (HIE) via a novel proteomic approach, the isobaric tags for absolute and relative quantification (iTRAQ) method.

Methods: Blood samples were collected from neonates with mild (n = 4), moderate (n = 4), or severe (n = 4) HIE who were admitted to the neonatal intensive care unit of Children's Hospital of Soochow University between Oct 2015 and Oct 2017. iTRAQ was performed in HIE patients and healthy controls (n = 4). Bioinformatics analyses including Gene Ontology and KEGG pathway enrichment analysis were performed to evaluate the potential features and capabilities of the identified differentially expressed proteins.

Results: A total of 51 commonly differentially expressed proteins were identified among the comparisons between mild, moderate, and severe HIE as well as healthy controls. Haptoglobin (HP) and S100A8 were most significantly up-regulated in patients with HIE and further validated via real-time PCR and western blotting. The differentially expressed proteins represented multiple biological processes, cellular components and molecular functions and were markedly enriched in complement and coagulation cascades.

Conclusions: HP and S100A8 may serve as a potential biomarker for neonatal HIE and reflects the severity of HIE. The complement and coagulation cascades play crucial roles in the development of neonatal HIE.

Keywords: Haptoglobin; Hypoxic-ischemic encephalopathy; Isobaric tags for absolute and relative quantification; Neonate; S100A8.

Fig. 1

Differentially expressed proteins among HIE patients and healthy controls. a, b Clustering heatmap and Volcano map of proteins differentially expressed between mild HIE and controls. c, d Clustering heatmap and Volcano map for the comparison of moderate HIE vs control. e, f Clustering heatmap and Volcano map for the comparison of severe HIE vs control. The green dots indicate down-regulated proteins, and the red dots indicate up-regulated proteins. The black dots depict the non-significant differentially expressed proteins

Fig. 2

Venn diagram depicting 51 commonly differentially expressed proteins in all three comparisons of mild HIE vs controls, moderate HIE vs controls, and severe HIE vs controls

Fig. 3

Overview of GO enrichment analysis and KEGG pathway analysis. a Differentially expressed proteins (mild HIE vs controls) were associated with major biological processes (BPs), cell localizations (CCs) and molecular functions (MFs). b Major pathways involved (mild HIE vs controls). c Summary of GO enrichment analysis and KEGG pathway analysis for mild HIE vs controls. d, e, f Features and participating pathways for differentially expressed proteins between moderate HIE vs controls. g, h, i Features and participating pathways of differentially expressed proteins between severe HIE vs controls

Fig. 4

qPCR and Western Blot analysis of HP and S100A8 expression. a, b Relative expression of S100A8 and HP in qPCR. c, d Relative expression of HP in Western Blot