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BRAIN Publication-derived

Cntnap2-Dependent Multi-Omics Analysis of Autism Spectrum Disorder: Integrated Proteometabolomic and Single-Cell RNA-Seq Protocol

Source Jang et al., 2023 · DGIST (Daegu Gyeongbuk Institute of Science and Technology) · 10.1038/s41380-022-01822-1

👤 Wooyoung Eric Jang, Ji Hwan Park, Gaeun Park, Geul Bang, Chan Hyun Na, Jin Young Kim, Kwang-Youl Kim, Kwang Pyo Kim, Chan Young Shin, Joon-Yong An, Yong-Seok Lee, Min-Sik Kim ⏱ 75 days 📋 5 phases 🧫 Patient-Derived iPSC (CNTNAP2-associated ASD); Mouse Neural Tissue (Cntnap2 KO)

Abstract

This protocol integrates quantitative mass spectrometry-based proteometabolomic analysis of medial prefrontal cortex tissue from Cntnap2 knockout mice with publicly available multi-omics data from autism spectrum disorder (ASD) patient brains and CNTNAP2-mutant forebrain organoids. The workflow identifies Cntnap2-dependent molecular networks dysregulated in ASD, revealing alterations in mitochondrial function, synaptic vesicle transport, and axonal integrity through integrated data analysis and functional network modeling.

Cell source
Patient-Derived iPSC (CNTNAP2-associated ASD); Mouse Neural Tissue (Cntnap2 KO)
Application
Disease modeling; Multi-omics biomarker discovery for autism spectrum disorder

Protocol overview

23 steps across 5 phases

Mouse Model Preparation and Tissue Dissection Day 1-14
  1. 1 Animal breeding and genotyping
  2. 2 Three-chamber social preference behavioral test
  3. 3 Medial prefrontal cortex tissue dissection
Protein Extraction and Quantitative Proteomic Analysis Day 15-30
  1. 1 Protein extraction from mPFC tissue
  2. 2 Enzymatic digestion with trypsin
  3. 3 TMT 10-plex labeling of peptides
  4. 4 Pool labeled peptides and reversed-phase liquid chromatography fractionation
  5. 5 High-resolution Orbitrap MS analysis in data-dependent acquisition mode
Targeted Quantitative Metabolomic Analysis Day 31-45
  1. 1 Metabolite extraction from mPFC tissue
  2. 2 Internal standard spiking and aliquoting
  3. 3 LC–MS/MS analysis of amino acids and biogenic amines (Aliquot 1)
  4. 4 FIA-MS/MS analysis of lipid species (Aliquot 2)
Differential Expression Analysis and Data Integration Day 46-60
  1. 1 Identification of differentially expressed peptides (DEPeptides) from proteomics data
  2. 2 Identification of differentially expressed metabolites (DEMs) from metabolomic data
  3. 3 Comparison with human ASD patient multi-omics datasets
  4. 4 Gene ontology (GO) and functional enrichment analysis
  5. 5 Single-cell RNA-seq reanalysis of CNTNAP2-mutant forebrain organoids
  6. 6 Cell-type-specific differential gene expression analysis
  7. 7 Protein-protein interaction network analysis
Molecular Network Model Construction Day 61-75
  1. 1 Selection of pathway-relevant molecules
  2. 2 Network model 1: Myelin sheath and mitochondrial dysfunction
  3. 3 Network model 2: Synaptic vesicle dysfunction
  4. 4 Network model 3: Neuronal projection and growth cone dynamics

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Jang et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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