Skip to content
← Back to browse
BRAIN Publication-derived

Generation and Proteomic Analysis of Schizophrenia Patient-Derived Cerebral Organoids

Source Notaras et al., 2021 · Center for Neurogenetics, Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, Cornell University · 10.1038/s41398-021-01664-5

👤 Michael Notaras, Aiman Lodhi, Haoyun Fang, David Greening, Dilek Colak ⏱ 40 days 📋 8 phases 🧫 Patient-Derived iPSC (Schizophrenia), Human iPSC (Control)

Abstract

This protocol describes the generation of 3D cerebral organoids from schizophrenia patient-derived and control human iPSCs, followed by comprehensive quantitative proteomics using TMTpro 16-plex isobaric barcoding and liquid chromatography-mass spectrometry. The procedure yields a proteomic map of developing brain tissue that reveals alterations in neuronal development factors and GWAS-implicated proteins in schizophrenia.

Cell source
Patient-Derived iPSC (Schizophrenia), Human iPSC (Control)
Application
Disease modeling and proteomic profiling

Protocol overview

35 steps across 8 phases

iPSC Culture and Maintenance Ongoing
  1. 1 Acquire iPSC lines
  2. 2 Culture iPSC colonies on vitronectin-coated plates
Embryoid Body Formation and Neural Induction Days 1-10 in vitro (DIV)
  1. 3 Dissociate iPSC colonies to single cells
  2. 4 Culture iPSC suspensions in ultra-low attachment plates to form embryoid bodies
  3. 5 Isolate healthy embryoid bodies and transfer to neural induction culture
  4. 6 Perform neural induction for up to 7 days
Organoid Maturation and Expansion Days 8-40 DIV
  1. 7 Embed organoids in Matrigel droplets
  2. 8 Culture organoids in terminal organoid media without agitation
  3. 9 Initiate orbital shaker agitation
  4. 10 Maintain organoids until harvest at 35-40 DIV
Sample Preparation for Proteomics Days 35-40 DIV (harvest) + same day
  1. 11 Harvest organoids and prepare for lysis
  2. 12 Lyse organoid tissue and extract protein
  3. 13 Reduce protein disulfide bonds
  4. 14 Alkylate cysteine residues
  5. 15 Perform enzymatic digestion with trypsin
Isobaric Barcoding and Sample Multiplexing Same day as digestion completion
  1. 16 Prepare peptide suspensions
  2. 17 Label samples with TMTpro 16-plex reagents
  3. 18 Quench unreacted TMT reagent
  4. 19 Pool labeled samples
Sample Desalting and Purification Same day as multiplexing
  1. 20 Desalt multiplexed peptide samples using C18 stage-tips
  2. 21 Dry desalted samples
Liquid Chromatography-Mass Spectrometry Analysis Days after sample preparation
  1. 22 Set up EASY-nLC 1200 liquid chromatography system
  2. 23 Perform peptide separation via gradient LC
  3. 24 Acquire full-scan mass spectra
  4. 25 Select and fragment precursor ions
  5. 26 Acquire tandem mass spectra (MS/MS) of fragment ions
Data Processing and Bioinformatics Post-analysis
  1. 27 Pre-process raw mass spectra
  2. 28 Perform peptide identification using MaxQuant
  3. 29 Filter identifications and exclude contaminants
  4. 30 Quantify proteins using TMT reporter ion intensity
  5. 31 Log2-transform and normalize protein intensities
  6. 32 Perform principal component analysis (PCA) and quality assessment
  7. 33 Impute missing values
  8. 34 Perform statistical testing for differential expression
  9. 35 Perform functional enrichment analysis

Full SOP

🔬

Create a free account to access this protocol

Join OrganMatch to unlock step-by-step procedures, reagent concentrations, QC checklists, and downloadable batch record templates.

Create free account

Already registered? Log in

Attribution

This SOP was authored by Organthis based on the published method in Notaras et al., 2021. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

Something wrong with this entry? Report an issue with this protocol

Need a commercial licence?
Use this protocol in your therapeutic or diagnostic pipeline.
Request Commercial Licence