Dec 23, 2024
Version 2
Neurogenesis with iron supplementation and label-free nDIA proteomics V.2
Version 1 is forked from Neural differentiation of AAVS1-TRE3G-NGN2 pluripotent stem cells
- Felix Kraus1,
- Yuchen He1,
- Harper JW1,2
- 1Department of Cell Biology, Blavatnik Institute, Harvard Medical School, 240 Longwood Ave, Boston MA 02115, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
External link: https://doi.org/10.7554/eLife.72328
Protocol Citation: Felix Kraus, Yuchen He, Harper JW 2024. Neurogenesis with iron supplementation and label-free nDIA proteomics. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwmydv8j/v2Version created by Felix Kraus
Manuscript citation:
Eapen VV, Swarup S, Hoyer MJ, Paulo JA, Harper JW, Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy. eLife doi: 10.7554/eLife.72328
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
We use this protocol and it's working
Created: December 23, 2024
Last Modified: December 23, 2024
Protocol Integer ID: 116879
Keywords: ASAPCRN, neurogenesis with iron supplementation, free ndia proteomic, free ndia proteomics this protocol, neurogenesi, free proteomic, h9 stem cell, differentiation of h9 stem cell, iron supplementation, ngn2
Funders Acknowledgements:
ASAP
Grant ID: ASAP Grant ID: ASAP-025160
ASAP
Grant ID: ASAP Grant ID: ASAP-000282
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Abstract
This protocol describes the NGN2-driven differentiation of H9 stem cells into iNeurons ± FAC iron supplementation. The protocol also covers sample preparation of label free proteomics and analysis via nDIA on the Astral Orbitrap mass analyzer.
Attachments
Troubleshooting
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Prepare ND1 Medium and ND2 Medium:
ND1 Medium
DMEM/F12
N2 (100x) 1x
BDNF 10 ng/ml
NT3 10 ng/ml
NEAA (100X) 1x
Laminin 0.2 μg/ml
Doxycycline 2 μg/ml
ND2 Medium
Neurobasal medium
B27 (50x) 1x
Glutamax (100x) 1x
BDNF 10 ng/ml
NT3 10 ng/ml
Doxycycline 2 μg/ml
Note
* Doxycycline can be withdrawn on Day 10 (or even earlier-not tested yet).
* Cells can be split from Day 5 to Day 7 (or even earlier-not tested yet).
Neuronal differentiation - Day 0:
Treat H9 AAVS1-TRE3G-NGN2 cells with Accutase and plate the dissociated cells in matrigel-coated 6-well plates (2x105 cells/well) in ND1 Medium supplemented with Y27632 (10 micromolar (µM) ).
Harvest samples for day 0. Wash wells twice in 1xPBS and harvest in ice-cold 1xPBS on ice. Snap freeze samples and store at -80°C.
Day 1:
Replace the medium with ND1 Medium.
For iron-addback samples, supplement media with 10µM FAC
Day 2:
Replace the medium with ND2 Medium.
For iron-addback samples, supplement media with 10µM FAC
Day 4:
Exchange 50% of the medium from each well.
For iron-addback samples, supplement media with 10µM FAC
Harvest samples for day 4. Wash wells twice in 1xPBS and harvest in ice-cold 1xPBS on ice. Snap freeze samples and store at -80°C.
Day 6:
Treat the cells with Accutase and replate the dissociated cells in matrigel-coated 6-well plates (2-4x105 cells/well) in ND2 Medium.
For iron-addback samples, supplement media with 10µM FAC
Day 8
Exchange 50% of the medium from each well every other day.
For iron-addback samples, supplement media with 10µM FAC
Note
* Doxycycline can be withdrawn on Day 10 (or even earlier-not tested yet).
* Cells can be split from Day 5 to Day 7 (or even earlier-not tested yet).
Harvest samples for day 8. Wash wells twice in 1xPBS and harvest in ice-cold 1xPBS on ice. Snap freeze samples and store at -80°C.
Day 10: Exchange 50% of the medium from each well.
For iron-addback samples, supplement media with 10µM FAC
Day 12: Exchange 50% of the medium from each well.
For iron-addback samples, supplement media with 10µM FAC
Day 14: Exchange 50% of the medium from each well.
For iron-addback samples, supplement media with 10µM FAC
Day 16
Day 16: Exchange 50% of the medium from each well.
For iron-addback samples, supplement media with 10µM FAC
Harvest samples for day 16. Wash wells twice in 1xPBS and harvest in ice-cold 1xPBS on ice. Snap freeze samples and store at -80°C.
Day 18: Exchange 50% of the medium from each well.
For iron-addback samples, supplement media with 10µM FAC
Day 20: Exchange 50% of the medium from each well.
For iron-addback samples, supplement media with 10µM FAC
Day 21
Harvest samples for day 21. Wash wells twice in 1xPBS and harvest in ice-cold 1xPBS on ice. Snap freeze samples and store at -80°C.
Sample Prep for LFQ
Cell pellets are lysed in 140 μL of 8 M Urea and 100 mM Tris (pH 8.0) using a syringe equipped with a 21G needle.
The protein concentration is measured using a protein BCA assay.
10 mM TCEP is added to the lysate for protein reduction.
40 mM chloroacetamide is added to the lysate for protein alkylation.
LysC (from FUJIFILM Wako) is added to the lysate at a protein-to-LysC ratio of 50:1.
The sample is incubated on a rocker for 4 hours at room temperature.
The urea concentration in the sample is diluted to 2 M by adding 100 mM Tris (pH 8.0).
Trypsin is added to the sample at a protein-to-trypsin ratio of 50:1.
The sample is incubated on a rocker overnight at room temperature to facilitate digestion.
Digested peptides are desalted using Waters 25 mg Sep-Pak tC18 96-well plates (Cat# 186002319).
The concentration of the desalted peptides is measured using a peptide BCA assay.
label-free nDIA proteomics
Separation is performed on a C18 capillary column (30 cm length × 100 μm inner diameter) packed with Accucore C18 resin (2.6 μm, 150 Å, Thermo Fisher Scientific) at a temperature of 55 °C using an EASY-nLC 1200 system (Thermo Scientific).
The flow rate is set to 450 nL/min.
Mobile phase A is prepared as 0.125% formic acid in ACN/water (5:95, v/v).
Mobile phase B is prepared as 0.125% formic acid in ACN/water (95:5, v/v).
Peptides are loaded onto the column, and the gradient of mobile phase B is increased from 5% to 23% over 22 minutes.
The gradient is further increased to 100% over 2 minutes and maintained at 100% for 6 minutes.
Eluting peptides are analyzed using an Orbitrap Astral mass spectrometer (Thermo Scientific).
Spray voltage is set to 2.2 kV.
The ion transfer tube temperature is maintained at 290 °C.
The FAIMS compensation voltage (CV) is set to -55 V.
The MS1 scan range is set from 380 to 980 m/z.
The MS1 resolution is set to 240,000 (at 200 m/z).
The source RF is configured at 50%.
The MS1 AGC target is set to 500%.
The MS1 injection time is set to 50 ms.
Precursors are isolated in the quadrupole using an isolation width of 2 m/z.
Fragmentation is performed by higher-energy collisional dissociation (HCD) with a normalized collision energy (NCE) of 27%.
MS2 mass spectra are acquired in data-independent mode using the Astral analyzer.
The MS2 scan range is set from 110 to 2,000 m/z.
The MS2 AGC target is configured at 500%.
The MS2 maximum injection time is set to 3 ms.
Raw files were converted to mzML format using MSConvert and analyzed using FragPipe (v22.0) with DIA-NN (v1.8.2). All settings were kept default.