Identifying Immune-related Molecular Biomarkers in Autism Spectrum Disorder Using Data-independent Acquisition Proteomics and Machine Learning

Summary

Here, we present a protocol using data-independent acquisition mass spectrometry and machine learning that identified eight immune-related proteins as accurate biomarkers for early Autism spectrum disorder diagnosis, validated by an enzyme-linked immunosorbent assay.

Abstract

This study presents a reproducible protocol for identifying serum protein biomarkers associated with autism spectrum disorder (ASD) using data-independent acquisition (DIA) mass spectrometry combined with machine learning (ML). DIA enables unbiased, high-resolution profiling of the serum proteome, including low-abundance proteins, while ensuring reproducibility across samples. ML approaches were applied to select diagnostically informative protein panels and improve model robustness. The analysis included serum from 99 children with ASD and 70 age-matched controls. High-abundance proteins were depleted, peptides were prepared using standardized digestion and fractionation procedures, and DIA was performed on a high-resolution mass spectrometer. Data processing and quantification identified differentially expressed proteins, which underwent functional enrichment analysis. Eight immune-related proteins emerged as strong candidates for biomarker development. A logistic regression model trained on these proteins achieved 95.27% accuracy, a Kappa value of 0.9025, and an AUC of 1.000 in cross-validation. These findings demonstrate the potential of DIA-based proteomics, combined with machine learning, as a robust framework for biomarker discovery in ASD and for adaptation in broader clinical research.

Introduction

Autism spectrum disorder (ASD) is a group of early-onset neurodevelopmental disorders characterized by heterogeneity in etiology and clinical presentation. Core features include persistent deficits in social communication and interaction, as well as restricted, repetitive behaviors, interests, or activities. In the United States, prevalence is approximately 2.3% among 8-year-old children and around 2.2% among adults, underscoring its public health impact^1,2,3,4. Risk factors are diverse, including genetic predispositions, immune dysregulatio....

Protocol

The protocol was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the Institutional Review Board at Changsha Maternal and Child Health Hospital; informed consent was obtained from the subjects.

1. Identification of children with autism with DSM-5

1. Collecting medical history and background information
   1. Developmental history
      1. Collect information on the patient's early development, including language, social, and motor skill progression.
      2. Note any developmental delays or abnormalities (e.g., language delay, difficulties in social interaction).

Results

The study included 99 children with ASD and 70 age-matched controls (3-7 years), with balanced sex distribution (Supplemental Table S2). Serum was collected after overnight fasting using standardized protocols: blood was drawn into serum separator tubes, allowed to clot at room temperature for 30 min, then centrifuged at 1,500 × g for 10 min at 4 °C. The supernatant was aliquoted and stored at −80 °C until further processing. High-abundance proteins (e.g., albumi.......

Discussion

The protocol described in this manuscript outlines a comprehensive approach for identifying immune-related molecular biomarkers in autism spectrum disorder (ASD) using data-independent acquisition (DIA) mass spectrometry and machine learning techniques. The important steps within the protocol ensure reliable and reproducible results, while also highlighting areas where modifications or troubleshooting may be necessary (Table 2).

One of the key steps in the protocol is the coll.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents and Chemicals	Acetonitrile (HPLC Grade)	Fisher Scientific	A18-50
Reagents and Chemicals	Ammonium bicarbonate (NH?HCO?)	Sigma-Aldrich	38939
Reagents and Chemicals	Ammonium formate	Sigma-Aldrich	90265
Reagents and Chemicals	Bovine Serum Albumin (BSA)	Thermo Fisher Scientific	23212
Reagents and Chemicals	Dithiothreitol (DTT)	Sigma-Aldrich	43815
Reagents and Chemicals	Formic acid (0.1%)	Thermo Fisher Scientific	28905
Reagents and Chemicals	Iodoacetamide (IAA)	Sigma-Aldrich	I1149
Reagents and Chemicals	Methanol (HPLC Grade)	Fisher Scientific	A452-4
Reagents and Chemicals	Trifluoroacetic acid (TFA)	Sigma-Aldrich	T6508
Reagents and Chemicals	Urea	Sigma-Aldrich	U5378
Kits and Specialized Reagents	BCA Protein Assay Kit	Thermo Fisher Scientific	23227
Kits and Specialized Reagents	C18 Sep-Pak Cartridges	Waters	WAT023590
Kits and Specialized Reagents	C18 StageTips (homemade)	3M Empore™
Kits and Specialized Reagents	High-Abundance Protein Depletion Kit	Millipore Sigma	122642
Kits and Specialized Reagents	iRT Standard Peptides	Biognosys AG
Kits and Specialized Reagents	Lysozyme ELISA Kit	Wuhan Fine Biotech Co.	Ltd.
Kits and Specialized Reagents	Trypsin/LysC Enzyme Mix	Promega	V5071
Equipment	Centrifuge	Eppendorf	5430R
Equipment	Easy-nLC 1200 System	Thermo Fisher Scientific
Equipment	Nanodrop One Spectrophotometer	Thermo Fisher Scientific	ND-ONE-W
Equipment	Q Exactive HF-X Mass Spectrometer	Thermo Fisher Scientific
Equipment	SpeedVac Concentrator	Thermo Fisher Scientific	SPD131DDA
Equipment	Swinning-Bucket Rotor Centrifuge	Various
Equipment	Waters XBridge BEH130 Column	Waters	C18, 3.5 μm, 2.1×150 mm
Equipment	Agilent 1260 HPLC System	Agilent	1260 Infinity II
Software and Online Tools	Bioconductor (R packages)	bioconductor.org
Software and Online Tools	caret (R package)	CRAN	caret_6.0-93
Software and Online Tools	clusterProfiler (R package)	Bioconductor	4.0.5
Software and Online Tools	DIA-NN	DIA-NN software	v1.8
Software and Online Tools	ggplot2 (R package)	CRAN	3.4.0
Software and Online Tools	MaxQuant	Max Planck Institute	1.6.17
Software and Online Tools	omickits.com	OmiKits Cloud Platform	http://www.omickits.com
Software and Online Tools	pROC (R package)	CRAN	1.18.0
Software and Online Tools	randomForest (R package)	CRAN	4.7-1.1
Software and Online Tools	Spectronaut Pulsar X	Biognosys AG	17
Software and Online Tools	UniProtKB Human Database	uniprot.org	Release 2019_10
Other Materials	Low-bind Microcentrifuge Tubes	Eppendorf	30120094
Other Materials	Serum Separator Tubes (SST)	BD Biosciences	367988
Other Materials	3M Empore™ C18 Disks	3M