Integrated Plasma Proteomic and Metabolic Profiling Reveals Signatures linked to Immune-Metabolic Dysregulation in Boys with Autism Spectrum Disorder

Abstract

Autism spectrum disorder (ASD) currently lacks validated blood-based biomarkers, and its precise etiology remains elusive. Our current study employed data-independent acquisition mass spectrometry (DIA-MS) to perform plasma proteomic profiling, with integrated metabolomic analysis, in 12 boys with ASD and 8 typically developing (TD) controls. A total of 453 proteins were quantified, of which 56 were identified as differentially expressed proteins (DEPs), including 27 up-regulated and 29 down-regulated in ASD. Functional enrichment analysis revealed that these DEPs are primarily involved in innate immune responses, complement activation, neutrophil extracellular trap formation, and coagulation cascades. Through random forest modeling with leave-one-out cross-validation (LOOCV) and receiver operating characteristic (ROC) analysis, we identified a signature of three proteins, including adipocyte plasma membrane-associated protein (APMAP), von Willebrand factor (VWF), and fibronectin 1 (FN1), as exploratory candidates,exhibiting an area under the curve (AUC) value of 0.906 in predicting ASD. Furthermore, integrated Orthogonal Two-way Partial Least Squares (O2PLS) analysis of the plasma proteome and metabolome uncovered potential immune-metabolic interactions, especially negative correlations between inflammation-related proteins, such as Lipopolysaccharide Binding Protein (LBP), Cluster of Differentiation 14 (CD14) and complement C4A (C4A), and metabolites such as cyclic ADP-ribose, alongside positive correlations centered on apolipoprotein C3 (APOC3) and antioxidant-related metabolites, such as selenocysteine. Moreover, strong correlations were observed between these DEPs and ASD behavioral features in boys with ASD. These findings suggest potential signatures of sustained immune activation, closely linked to systemic metabolic dysregulation in ASD.