Bulk Proteomics (DIA-MS) of Human Dorsal Root Ganglion

Allison Barry; Theodore Price; Manuela Schmidt

Oct 24, 2024

Bulk Proteomics (DIA-MS) of Human Dorsal Root Ganglion

DOI

dx.doi.org/10.17504/protocols.io.j8nlk8j56l5r/v1

Allison Barry^1,2,
Theodore Price¹,
Manuela Schmidt²

¹University of Texas Dallas;
²University of Vienna

PRECISION Human Pain Network

Allison Barry

University of Texas Dallas

DOI: dx.doi.org/10.17504/protocols.io.j8nlk8j56l5r/v1

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

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

 
EquipmentCompanyLocation
LoBind Protein Eppendorf tube EppendorfHamburg, DE
Bioruptor Pico Diagenode SeraingBelgium
ThermoMixer C EppendorfHamburg, DE
NanoPhotometer N60ImplenMunich, DE
DynaMag™-2 MagnetThermoFisher
timsTOF HTBrukerBremen, DE
C18 trap column (1 mm x 5 mm)ThermoFisher
AuroraTM ULTIMATE column (25 cm × 75 µm)IonOpticksAustralia
Table: Required equipment.

ComponentProviderIdentifierCompany RRID
Tris 1 MAccugene/AvantorCAT# 733-1653RRID:SCR_000377
Sera-Mag SpeedBead beadsCytivaCAT# 65152105050250, CAT# 45152105050250 at a 1:1 mixRRID:SCR_023581
10× PBSFisher ScientificCAT# 11594516RRID:SCR_008452
AcetonitrileFisher ScientificCAT# 10001334RRID:SCR_008452
Formic acidFisher ScientificCAT# 15658430RRID:SCR_008452
GlycerolFisher ScientificCAT# 10021083RRID:SCR_008452
Trypsin/Lys-CPromegaCAT# V5073RRID:SCR_006724
AcetoneSigma-AldrichCAT# 1000201000RRID:SCR_008988
Ammonium bicarbonateSigma-AldrichCAT# 09830-500GRRID:SCR_008988
Dithiothreitol (DTT) 1 MSigma-AldrichCAT# 43816RRID:SCR_008988
EthanolSigma-AldrichCAT# 1117272500RRID:SCR_008988
IodoacetamideSigma-AldrichCAT# I1149RRID:SCR_008988
Water MS gradeSigma-AldrichCAT# 1.15333.1000RRID:SCR_008988
Table: Required Chemicals.
 
SoftwareVersionCitationRRID
R>4.2https://www.r-project.org/RRID:SCR_001905
Bioconductorhttp://www.bioconductor.org/ 

RRID:SCR_006442
limmaRitchie et al., 2015RRID:SCR_010943
clusterProfilerWu et al., 2021RRID:SCR_016884
DIA-NN1.8.1Demichev et al., 2020RRID:SCR_022865
Table: General software for processing

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 
Fig 1. Dissection schematic

Fig 2. Photographs of clean hDRG. A. DRG before (left) and after (right) transecting the nerve root (NR) from the ganglia (G). B. Low magnification photo of the pigmentation aggregates visible within the ganglia (view down dissecting microscope).

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

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

Collect beads on a magnetic rack (2 min) and discard the supernatant

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).

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

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

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

Public workspaceBulk Proteomics (DIA-MS) of Human Dorsal Root Ganglion

Bulk Proteomics (DIA-MS) of Human Dorsal Root Ganglion