Sep 17, 2020

Public workspaceImmunoprecipitation (IP) and Mass Spectrometry

This protocol is a draft, published without a DOI.
  • Yingchao Xue1,2,
  • Xiping Zhan3,
  • Shisheng Sun4,
  • Senthilkumar S. Karuppagounder5,6,7,
  • Shuli Xia2,5,
  • Valina L Dawson5,6,7,8,9,
  • Ted M Dawson5,6,7,8,10,
  • John Laterra2,5,8,11,
  • Jianmin Zhang1,
  • Mingyao Ying2,5
  • 1Department of Immunology, Research Center on Pediatric Development and Diseases, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, State Key Laboratory of Medical Molecular Biology;
  • 2Hugo W. Moser Research Institute at Kennedy Krieger;
  • 3Department of Physiology and Biophysics, Howard University;
  • 4College of Life Sciences, Northwest University;
  • 5Department of Neurology, Johns Hopkins University School of Medicine;
  • 6Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine;
  • 7Adrienne Helis Malvin Medical Research Foundation;
  • 8Department of Neuroscience, Johns Hopkins University School of Medicine;
  • 9Department of Physiology, Johns Hopkins University School of Medicine;
  • 10Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine;
  • 11Department of Oncology, Johns Hopkins University School of Medicine
  • Neurodegeneration Method Development Community
  • Metabolomics Protocols & Workflows
Anita Broellochs
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External link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344911/
Protocol Citation: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying 2020. Immunoprecipitation (IP) and Mass Spectrometry. protocols.io https://protocols.io/view/immunoprecipitation-ip-and-mass-spectrometry-9zhh736
Manuscript citation:
Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. Xue Y, Zhan X, Sun S, Karuppagounder SS, Xia S, Dawson VL, Dawson TM, Laterra J, Zhang J, Ying M. Stem Cells Transl Med. 2019 Feb;8(2):112-123. doi: 10.1002/sctm.18-0036. Epub 2018 Nov 1. PMID: 30387318
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
These protocols were published in: Xue Y, Zhan X, Sun S, et al. Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons. Stem Cells Transl Med. 2019;8(2):112–123. doi:10.1002/sctm.18-0036
Created: December 02, 2019
Last Modified: September 17, 2020
Protocol Integer ID: 30473
Keywords: ND1014, N1, ND27760, ipsc, SNCA, Atoh2, Ngn2, Immunoprecipitation, IP, Mass Spectrometry, MS
Abstract
This protocol explains Immunoprecipitation (IP) and Mass Spectrometry for lines ND1014, N1, and ND27760 from Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons.
Materials
MATERIALS
ReagentIodoacetamideP212121Catalog #AK-U470
ReagentSodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
ReagentSequencing Grade Modified Trypsin, 100ugPromegaCatalog #V5117
ReagentTCEP-HClGold BiotechnologyCatalog #TCEP
ReagentUreaMerck MilliporeSigma (Sigma-Aldrich)Catalog #U5378
ReagentNE-PER Nuclear and Cytoplasmic Extraction ReagentsThermo ScientificCatalog #78833
ReagentMonoclonal ANTI-FLAG M2 antibody produced in mouseMerck MilliporeSigma (Sigma-Aldrich)Catalog #F3165
ReagentDynabeads Protein G for ImmunoprecipitationThermo ScientificCatalog #10003D
Safety warnings
Please refer to the Safety Data Sheets (SDS) for safety and environmental hazards.
Before start
Obtain approval to work with human stem cells from an appropriate Institutional Review Board.
Immunoprecipitation (IP)
Immunoprecipitation (IP)
Twenty-four hours after mRNA transfection, extract nuclear proteins of iPSCs using the NE-PER Nuclear and Cytoplasmic Extraction Kit.
Perform FLAG IP using anti-FLAG M2 antibody and Protein G Dynabeads following the manufacturer’s protocol.
Elute binding proteins by elution buffer (Concentration8 Molarity (M) urea , Concentration1 Molarity (M) NaHCO3 in water ).

Mass Spectrometry (MS)
Mass Spectrometry (MS)
Denature protein samples in Concentration8 Molarity (M) urea /Concentration1 Molarity (M) NH4HCO3 buffer .

Reduce protein samples by Concentration10 millimolar (mM) TCEP at Temperature37 °C for Duration01:00:00 .

Incubation
Alkylate protein samples by Concentration15 millimolar (mM) iodoacetamide at TemperatureRoom temperature in the dark for Duration00:30:00 .

Incubation
Dilute samples fivefold with deionized water.
Add sequencing grade trypsin (50:1, w/w) to the samples.
Incubate at Temperature37 °C DurationOvernight with shaking.

Incubation
Centrifuge at Centrifigation13000 x g, 00:10:00 to remove any particulate matter.

Centrifigation
Purify by a C18 solid-phase extraction.
Elute peptides from the C18 column in 60% ACN/0.1% TFA.
Measure peptide concentrations using a NanoDrop microvolume spectrophotometer.
Resuspend in 0.2% FA solution.
Inject 1 μg in 6 μl solution for each LC–MS/MS analysis using Velos Orbitrap mass spectrometer with a 120-minute online LC separation.
IP and MS Analysis
IP and MS Analysis
Search raw data against UniProt human protein database by Proteome Discoverer with the following parameters:
  • two missed cleavages allowed for trypsin digestion
  • carbamidomethyl (C) set as a fixed modification
  • oxidation (M) and acetyl (protein N-terminal) set as variable modifications
Normalize the intensity of each identified proteins to the intensity of their A-WT or A-SA proteins, respectively, and further calculate the A-SA/A-WT ratio of normalized intensity.
Computational step