Oct 24, 2024
Bulk Proteomics (DIA-MS) of Human Dorsal Root Ganglion
- Allison Barry1,2,
- Theodore Price1,
- Manuela Schmidt2
- 1University of Texas Dallas;
- 2University of Vienna
- PRECISION Human Pain Network
Protocol Citation: Allison Barry, Theodore Price, Manuela Schmidt 2024. Bulk Proteomics (DIA-MS) of Human Dorsal Root Ganglion . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8j56l5r/v1
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: September 17, 2024
Last Modified: October 24, 2024
Protocol Integer ID: 107749
Keywords: DRG, sensory neurons, proteomics, DIA, mass spectrometry, bulk proteomics on frozen human dorsal root ganglia, bulk proteomic, frozen human dorsal root ganglia, human dorsal root ganglion, dorsal root ganglion, independent acquisition mass spectrometry, tissue preparation to data, mass spectrometry, tissue preparation, tissue from donor, human dorsal root ganglia
Abstract
In this protocol, we describe how to perform bulk proteomics on frozen human dorsal root ganglia (hDRG) tissue from donors from tissue preparation to data-independent acquisition mass spectrometry (DIA-MS).
Materials
| Equipment | Company | Location | |
| LoBind Protein Eppendorf tube | Eppendorf | Hamburg, DE | |
| Bioruptor Pico | Diagenode Seraing | Belgium | |
| ThermoMixer C | Eppendorf | Hamburg, DE | |
| NanoPhotometer N60 | Implen | Munich, DE | |
| DynaMag™-2 Magnet | ThermoFisher | ||
| timsTOF HT | Bruker | Bremen, DE | |
| C18 trap column (1 mm x 5 mm) | ThermoFisher | ||
| AuroraTM ULTIMATE column (25 cm × 75 µm) | IonOpticks | Australia |
Table: Required equipment.
| Component | Provider | Identifier | Company RRID | |
| Tris 1 M | Accugene/Avantor | CAT# 733-1653 | RRID:SCR_000377 | |
| Sera-Mag SpeedBead beads | Cytiva | CAT# 65152105050250, CAT# 45152105050250 at a 1:1 mix | RRID:SCR_023581 | |
| 10× PBS | Fisher Scientific | CAT# 11594516 | RRID:SCR_008452 | |
| Acetonitrile | Fisher Scientific | CAT# 10001334 | RRID:SCR_008452 | |
| Formic acid | Fisher Scientific | CAT# 15658430 | RRID:SCR_008452 | |
| Glycerol | Fisher Scientific | CAT# 10021083 | RRID:SCR_008452 | |
| Trypsin/Lys-C | Promega | CAT# V5073 | RRID:SCR_006724 | |
| Acetone | Sigma-Aldrich | CAT# 1000201000 | RRID:SCR_008988 | |
| Ammonium bicarbonate | Sigma-Aldrich | CAT# 09830-500G | RRID:SCR_008988 | |
| Dithiothreitol (DTT) 1 M | Sigma-Aldrich | CAT# 43816 | RRID:SCR_008988 | |
| Ethanol | Sigma-Aldrich | CAT# 1117272500 | RRID:SCR_008988 | |
| Iodoacetamide | Sigma-Aldrich | CAT# I1149 | RRID:SCR_008988 | |
| Water MS grade | Sigma-Aldrich | CAT# 1.15333.1000 | RRID:SCR_008988 |
Table: Required Chemicals.
| Software | Version | Citation | RRID | |
| R | >4.2 | https://www.r-project.org/ | RRID:SCR_001905 | |
| Bioconductor | http://www.bioconductor.org/ | RRID:SCR_006442 | ||
| limma | Ritchie et al., 2015 | RRID:SCR_010943 | ||
| clusterProfiler | Wu et al., 2021 | RRID:SCR_016884 | ||
| DIA-NN | 1.8.1 | Demichev et al., 2020 | RRID:SCR_022865 |
Table: General software for processing
Troubleshooting
Before start
Appropriate PPE includes lab coat and gloves. Proper safety precautions for work with and disposing off human tissue must be followed. Dissection tools should be cleaned and sterilized prior to starting.
Ensure you have all materials listed in the "Materials" section.
Tissue Harvesting
Extended tissue procurement methods are described in dx.doi.org/10.17504/protocols.io.kqdg32qr1v25/v1, see notes on fresh-frozen samples from steps 1-35
Surgically excised lumbar (L1-L5) human dorsal root ganglia (hDRGs) are obtained from organ donors at Southwest Transplant Alliance (STA) within 4h post cross-clamp
Each hDRG is placed in an epitube, labelled, and buried directly in powdered dry ice prior to transport (on dry ice) to a -80°C for storage
Here, tissue was then shipped on dry ice to another facility for proteome-specific processing, and again stored at -80°C
Tissue cleaning and lysis
15h 20m
12-24 hours prior to dissection, tissue is transferred to -20°C
12h
Chill chemicals: chill 100% acetone at -20°C and 80% EtOH at 4°C for later use
Prepare lysis buffer: 2% SDS, 100mM Tris, 5% glycerol, 10mM DTT and 1x protease inhibitor cocktail
Add 500 μl lysis buffer to LoBind Protein Eppendorf tubes, with 1-4 tubes per sample depending on tissue amount
For each DRG, tissue is thawn on ice for ~15 min prior to dissection (also on ice).
Rinse 1x with cold PBS and dissected to remove the surrounding connective tissue and fat. In each ganglia, a nerve root extending from the core ganglia was identified. This is presumed to contain fewer/no neuronal somata and is transected crudely with a scalpel for separate processing
15m
Due to the size of the tissue each hDRG can be cut into up multiple pieces (typically 1-4) and subsequently, each piece is cut in smaller pieces to increase the surface area for lysis. All small pieces of one big cut can then be transferred to a pre-filled eppendorf tube (step 5)
Sonicate tissue for 15 min (cycles of 30 sec ON and 30 sec OFF, 4°C, low frequency) in a Bioruptor Pico
15m
Incubate for 15 min at 70°C, 1500 rpm in a ThermoMixer C
15m
Centrifuge for 5 min with 10000 × g at room temperature (RT) to pellet cell debris
5m
Combine supernatant (~480 μl) with 5x sample volume 100% acetone (pre-chilled) and incubate at -20°C for 2.15-2.45 h to precipitate the proteins.
NOTE: Supernatant can be split into 2 eppendorfs per sample if there are volume constraints
2h 30m
Centrifuge for 30 min at 14000 × g, RT
30m
Remove supernatant and wash pellet with ice-cold 80% EtOH
Centrifuge for 30 min at 14000 × g, RT
30m
Remove EtOH and air dry pellets for 20 min
20m
Resolubilise pellet in lysis buffer by incubation for 10 min at 70°C, 1500 rpm (ThermoMixer C)
10m
Measure total protein concentration per eppendorf at 280 nm with a NanoPhotometer N60
If an hDRG was processed in multiple tubes due to the size, these can be merged by equal volume into a single sample. If combined, measure final concentration again.
Samples can now be flash-frozen and stored at -20°C
Protein clean-up and digestion
Perform as described in Xian et al., 2022, protein clean-up and digestion is based on the single-pot, solid-phase-enhanced sample preparation (SP3) method from Hughes et al. (Hughes et al., 2019). NOTE: Hughes et al., also contains a supplemental video outlining each SP3 step.
Reduction: Combine 50 µg total protein of each sample with 5 mM final concentration dithiothreitol (DTT) from a 1M stock for 30 min at 60°C
30m
Alkylation: mix with 20 mM iodoacetamide (IAA) for 30 min in the dark at RT
30m
Dilute 1M DTT stock to a final concentration of 5 mM to quench any remaining DTT for 15 min at RT
15m
Add 10 µL of pre-mixed Sera-Mag SpeedBead beads (1:1) to each sample, as well as one sample volume of absolute EtOH to initiate protein binding to the beads
Incubate in a ThermoMixer C for 5 min with 1000 rpm agitation at 24°C
5m
Collect beads on a magnetic rack (2 min) and discard the supernatant
2m
Rinse beads 3x with 500 μL of 80% EtOH
Reconstitute beads in 50 μL ammonium bicarbonate (50 mM, pH 8) containing 2 μg Trypsin/Lys-C. Incubate for 18 h at 37°C with 950 rpm agitation to digest proteins
18h
Peptide clean-up: acetonitrile (ACN) was added to each sample to a final concentration of 95%. Samples were incubated for 8 min at RT and then the beads were collected on a magnetic rack (2 min)
10m
Peptide clean-up: Discard the supernatant and wash beads with 900 μL of 100% ACN
Peptide elution: add 40 μL LC-MS grade H2O
Measure peptide concentration at 205 nm with a NanoPhotometer N60 (Implen)
Add formic acid (FA) to a final concentration of 0.1% and store at -20°C until further use
LC-MS/MS
Perform peptide analysis via NanoElute LC (Nanoflow reversed-phase liquid chromatography (Nano-RPLC) performed on a NanoElute2 system) coupled with a hybrid TIMS quadrupole TOF mass spectrometer (timsTOF HT, Bruker Daltonik) via a CaptiveSpray ion source. The following parameters are used:
Mobile phase A: 100% water, 0.1% FA.
Mobile phase B: 100% acetonitrile (ACN), 0.1% FA
500 ng of peptides
Load peptides onto C18 trap column (1 mm x 5 mm, ThermoFisher), further separating over a 90 min gradient on an AuroraTM ULTIMATE column (25 cm × 75 µm) packed with 1.6 µm C18 particles (IonOpticks, Fitzroy, Australia).
Flow rate = 250 nL/min, except for the last 7 minutes, where the flow rate accelerates to 400 nL/min
The mobile phase B increases linearly from 2 to 20% in the first 60 minutes, followed by another linear increase to 35% within 22 minutes and a steep increase to 85% in 0.5 min. Then, a flow rate switches to 400 nL/min in 0.5 min and is maintained for 7 minutes to the end of the gradient to elute all hydrophobic peptides.
Analyze samples in data-independent acquisition (DIA) mode coupled with parallel accumulation serial fragmentation (PASEF). Precursors with m/z between 350 and 1200 are defined in 13 cycles (either 2 or 3 quadrupole switches per cycle) containing 34 ion mobility steps within the ion mobility range of 0.65 – 1.35 (1/k0) with fixed isolation window of 25 Th in each step.
The acquisition time of each DIA-PASEF scan is set to 100 ms, resulting in a total cycle time of around 1.48 sec.
The collision energy is ramped linearly from 65 eV at 1/k0 = 1.6 to 20 eV at 1/k0 = 0.6.
DIA-PASEF data analysis
DIA-NN v1.8.1 (Demichev et a., 2020) is run with a predicted library search to process raw output files (`.d`) against the human proteome (UP000005640) with match-between-runs (MBR) enabled
#!/bin/bash
#SBATCH -J diann_hdrg
#SBATCH -N 1
#SBATCH --cpus-per-task=128
module purge
DATA=`\path\to\data\directory`
OUT=`\path\to\output\directory`
./bin/diann-1.8.1 --dir $DATA \
--gen-spec-lib --fasta-search --use-quant \
--predictor \
--fasta hdrg/UP000005640_validated.fasta \
--threads 128 --verbose 1 --qvalue 0.01 --matrices \
--min-corr 2.0 --corr-diff 1.0 --time-corr-only \
--min-fr-mz 100 --max-fr-mz 1700 \
--out $OUT/report.tsv \
--out-lib $OUT/report-lib.tsv \
--cut K*,R* --missed-cleavages 2 --min-pep-len 5 --max-pep-len 52 \
--min-pr-mz 350 --max-pr-mz 1200 --min-pr-charge 2 --max-pr-charge 4 \
--var-mods 3 --var-mod UniMod:35,15.994915,M --var-mod UniMod:1,42.010565,*n \
--monitor-mod UniMod:1 --rt-profiling --reanalyse --met-excision \
--pg-level 1 --no-ifs-removal
MaxLFQ per peptide and gene group is calculated in R using the diann package:
library(diann)
library(dplyr)
df <- diann_load("./report.tsv")
precursors <- diann_matrix(df, q = 0.01)
peptides.maxlfq <- diann_maxlfq(df[df$Q.Value <= 0.01 & df$PG.Q.Value <= 0.01,],
sample.header = "Run",
group.header="Stripped.Sequence",
id.header = "Precursor.Id",
quantity.header = "Precursor.Normalised")
gene.groups <- diann_maxlfq(df[df$Q.Value <= 0.01 & df$GG.Q.Value <= 0.01,],
sample.header = "Run",
group.header="Genes",
id.header = "Precursor.Id",
quantity.header = "Precursor.Normalised")
Downstream processing can then be performed on a matrix of gene group expression (eg. GSEA via clusterProfiler, differential expression testing with limma)
Protocol references
Demichev, V., Messner, C.B., Vernardis, S.I. et al. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat Methods 17, 41–44 (2020). https://doi.org/10.1038/s41592-019-0638-x
Hughes CS, Moggridge S, Müller T, Sorensen PH, Morin GB, Krijgsveld J. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc. 2019 Jan;14(1):68-85. doi: 10.1038/s41596-018-0082-x. PMID: 30464214.
Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015 Apr 20;43(7):e47. doi: 10.1093/nar/gkv007. Epub 2015 Jan 20. PMID: 25605792; PMCID: PMC4402510.
Xian, F., Sondermann, J.R., Gomez Varela, D., Schmidt, M. Deep proteome profiling reveals signatures of age and sex differences in paw skin and sciatic nerve of naïve mice. eLife 11:e81431 (2022) https://doi.org/10.7554/eLife.81431
Wu, Tianzhi, et al. "clusterProfiler 4.0: A universal enrichment tool for interpreting omics data." The innovation 2.3 (2021).