Jan 06, 2025
Proteomics Sample Preparation of Human Skin Punch Biopsies for Data-Independent Acquisition Mass Spectrometry Analysis
- Matthew Jaconelli1,
- Francesca Tonelli1,
- Dario Alessi1
- 1MRC-PPU, University of Dundee
Protocol Citation: Matthew Jaconelli, Francesca Tonelli, Dario Alessi 2025. Proteomics Sample Preparation of Human Skin Punch Biopsies for Data-Independent Acquisition Mass Spectrometry Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn9e46l5d/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: December 06, 2024
Last Modified: January 06, 2025
Protocol Integer ID: 117727
Keywords: ASAPCRN
Funders Acknowledgements:
Michael J Fox Foundation for Parkinson's Research
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Abstract
Protocol for the processing of human skin tissue punch biopsies from the C8 paravertebral region for data-independent acquisition mass spectrometry (DIA-MS) proteomics, including generation of a human skin proteome MS spectral library. This protocol was generated as part of a pilot study in collaboration with the Michael J. Fox Foundation. The biospecimens were obtained from the Parkinson’s Progression Marker Initiative (PPMI) (RRID:SCR_006431). For up-to-date information on the study, visit www.ppmi-info.org.
Materials
Acetonitrile ≥99.9%VWR International (Avantor)Catalog #1.00030.2500
Formic Acid, 99.0+%, Optima™ LC/MS Grade ThermoFisherScientific Catalog#A117-50
LC-grade WaterFisher ScientificCatalog #10777404
Ammonium formateMerck MilliporeSigma (Sigma-Aldrich)Catalog #70221-25G-F
Thermo Scientific™ EPA Screw Vial Assembled Kit, Vials/Septa/CapsThermo ScientificCatalog #11543750
Precellys Lysing Kit Catalog#P000922-LYSK0-A
Sodium Dodecyl Sulfate (SDS), Micropellets, Fisher BioReagentsThermo Fisher ScientificCatalog #15450685
HEPESFormediumCatalog #HEPES10
Bond-Breaker™ TCEP Solution, Neutral pHThermo Fisher ScientificCatalog #77720
IodoacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I1149-25G
Trifluoroacetic acid for HPLC > 99.0%Merck MilliporeSigma (Sigma-Aldrich)Catalog #302031-100ML
Methanol, for HPLC-MS, Fisher Chemical™Thermo Fisher ScientificCatalog #10653963
Triethylammonium bicarbonate bufferMerck MilliporeSigma (Sigma-Aldrich)Catalog #T7408-500ML
Thermo Scientific™ Mass Spectrometry Grade ProteasesThermo Fisher ScientificCatalog #15956915
n-Dodecyl-beta-Maltoside DetergentThermo FisherCatalog #89902
S-Trap™ Micro Column (≤ 100 µg)ProtifiCatalog #C02-micro-80
cOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #11873580001
Roche PhosSTOP™Merck MilliporeSigma (Sigma-Aldrich)Catalog #4906837001
Eppendorf™ twin.tec™ PCR Plates LoBind™ - PCR PlatesThermo Fisher ScientificCatalog #15280735
Pierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225
SafeSeal reaction tube, 2 ml, PP, PCR Performance Tested, Low protein-bindingSarstedtCatalog #72.695.600
Acclaim™ PepMap™ 100 C18 LC Columns, 3µm, 20mm length, 0.075mm I.D.Thermo FisherCatalog #164535
Thermo Scientific™ EASY-Spray™ PepMap™ Neo UHPLC ColumnsThermo ScientificCatalog #17477583
XBridge BEH C18 Column 130Å 3.5 µm 4.6 mm X 250 mm 1/pkWatersCatalog #186003943
Note
All solvents and buffers were prepared in or aliquoted from glass amber vials, using sterile (not autoclaved) pipette tips or glass cylinders.
Equipment/Software:
Vanquish Neo nano-liquid chromatography system (ThermoScientific)
Orbitrap Astral Mass Spectrometer (ThermoScientific)
Vanquish liquid chromatography system inline with fraction collector module (ThermoScientific)
ThermoMixer C (Eppendorf)
Savant SpeedVac SPD140DDA Vacuum Concentrator (ThermoScientific)
Micro Star 17 microcentrifuge (VWR)
Centurion Scientific Limited centrifuge
Precellys Evolution tissue homogeniser with Cryolys Evolution (Bertin Technologies)
BioRuptor Plus with minichiller 300 (Diagenode)
Xcalibur (ThermoScientific)
DIA-NN (version 1.8.1)
Protein Extraction
Protein Extraction
27m
27m
Transfer frozen skin tissue to a Precellys Lysing Kit (Bertin Technologies) 2ml ceramic bead tube containing 500 µL SDS lysis buffer: 2% SDS (w/v), 20 millimolar (mM) HEPES 8 , with complete EDTA-free protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail tablets (Roche).
Note
Place samples On ice immediately following transfer.
Immediately transfer samples to the Precellys Evolution tissue homogeniser (Bertin Technologies). Lyse tissue at 0 °C for 15x 30 second cycles at 6800 rpm , with 30 second rest between each cycle.
Centrifuge samples at 2000 x g, 4°C, 00:02:00 to reduce bubbles and transfer supernatant to fresh Protein LoBind Eppendorf tubes (Sarstedt).
2m
Heat samples at 95 °C for 00:05:00 on a heat-block (Grant, QBT2).
5m
Sonicate samples using a BioRuptor Diagenode (Diagenode) at 4 °C for 15x 30 second cycles, with 30 seconds rest between each cycle.
Centrifuge samples at 17000 x g, 4°C, 00:20:00 and carefully transfer supernatant to fresh Protein LoBind Eppendorf tubes (Sarstedt), taking care not to disturb the pelleted cell debris or any visible lipids.
20m
Quantify protein concentrations using BCA assay (ThermoScientific).
Note
Samples can be stored at -80 °C at this stage.
Aliquot 20 µg protein from each sample for total proteomic analysis.
Total Proteomics Sample Preparation
Total Proteomics Sample Preparation
1h
1h
Adjust total volume to 40 µL using SDS lysis buffer.
Reduce samples with 10 millimolar (mM) TCEP (final concentration, 100 millimolar (mM) TCEP stock in 300 millimolar (mM) TEABC) for 1100 rpm, 60°C, 00:30:00 (ThermoMixer C, Eppendorf).
30m
Alkylate samples in the dark with 40 millimolar (mM) iodoacetamide (final concentration, 400mM stock) for 1100 rpm, 25°C, 00:30:00 (ThermoMixer C, Eppendorf).
30m
Add 20% SDS to achieve a final concentration of 5% SDS, then acidify samples through addition of Trifluoracetic acid to a final concentration of 1%.
Micro S-Trap On-Column Digest
Micro S-Trap On-Column Digest
1h 22m
1h 22m
Add 6x sample volume of wash buffer (90% methanol, 10% 100mM TEABC).
Mix sample by pipetting up and down, then load 150 µL of sample onto micro-columns inside fresh 2ml Protein LoBind Eppendorf tubes (Sarstedt) within a centrifuge.
Centrifuge at 1000 x g, 00:01:00 and discard flow-through.
1m
Repeat go to step #15 until all sample has been loaded through micro-column.
Wash S-Trap columns with bound protein by adding 150 µL wash buffer followed by centrifugation at 1000 x g, 00:01:00 . Discard flow-through.
1m
Repeat step 17 three times.
Note
Ensure there is no remaining wash buffer in the S-trap micro columns before proceeding to step 19, as this may impact digestion efficiency.
Digest protein by addition of 60 µL (1.5μg) of Trypsin/Lys-C mix (MS grade, Promega, UK) in 50 millimolar (mM) TEABC solution (8 ) at 47 °C for 01:20:00 , followed by incubation at 22 °C Overnight (approximately 16 hours).
1h 20m
Peptide Elution
Peptide Elution
4m
4m
Elute samples by addition of 40 µL 50 millimolar (mM) TEABC (8 ) and centrifuge at 1000 x g, 00:01:00 .
Note
Do NOT discard flow-through containing peptides at any step of elution.
1m
Add 40 µL 0.15% (v/v) formic acid (FA) and centrifuge at 1000 x g, 00:01:00 .
1m
Add 40 µL 80% acetonitrile (ACN), 0.15% FA and centrifuge at 1000 x g, 00:01:00 . Repeat twice (3x total).
1m
Dry peptides at Room temperature using a vacuum centrifuge and store dried peptides at -20 °C until mass spectrometry analysis.
Peptide Resuspension
Peptide Resuspension
30m
30m
Resuspend samples by addition of 100 µL 0.1% formic acid supplemented with 0.015% N-Dodecyl-B-D-Maltoside (DDM) and mix at 1800 rpm, Room temperature , 00:30:00 (ThermoMixer C, Eppendorf).
30m
If analysing immediately, transfer 20 µL of each sample to a 96-well plate or LC-MS vials and inject 200 ng peptides per sample for MS analysis.
Offline HPLC Fractionation of Pooled Sample Peptides for Spectral Library Generation
Offline HPLC Fractionation of Pooled Sample Peptides for Spectral Library Generation
Aliquot 20 µL from each sample and pool in a 2 mL Protein LoBind Eppendorf tube (Sarstedt).
Note
Depending on sample numbers, this volume may need to be increased or decreased.
Transfer to an LC-MS vial/plate and inject 50% of pooled peptides (approximately 44 µg ) into a Vanquish HPLC system inline with a Fraction Collector using system settings.
Solvent A – 10mM Ammonium Formate (10 )
Solvent B – 10mM Ammonium Formate (10 ), 80% Acetonitrile
All changes are linear: Curve = 5
New fraction collected every 30 seconds starting at 21 minutes, directly into a LoBind twintec 96-well plate (Eppendorf).
Following fraction collection, vacuum centrifuge samples at Room temperature until dry and store at -20 °C until mass spectrometry analysis.
Note
Samples are dried and later resuspended in the original 96-well LoBind collection plate to minimise peptide losses through sample transfer, and all fractions are additionally subsequently injected directly from each well for MS analysis. Ensure plate is tightly sealed before storing at -20 °C .
Spectral Library Generation using Narrow-Window Data-Independent Acquisition Mass Spectrometry
Spectral Library Generation using Narrow-Window Data-Independent Acquisition Mass Spectrometry
32m
32m
Centrifuge dried peptides (contained within LoBind 96-well plate) for 54 rcf, 00:01:00 (Centurion Scientific).
1m
Add 20 µL of 0.1% formic acid supplemented with 0.015% N-Dodecyl-B-D-Maltoside (DDM) and mix at 500 rpm, Room temperature , 00:30:00 (ThermoMixer C, Eppendorf).
Note
To prevent potential sample loss, following addition of resuspension buffer, reseal the LoBind plate prior to shaking.
30m
Centrifuge resuspended peptides for54 rcf, 00:01:00 (Centurion Scientific). Remove plate seal.
1m
Inject 10 µL of each fraction into a Vanquish Neo UHPLC system inline with an Orbitrap Astral mass spectrometer for DIA-MS analysis.
Trap and elute peptides using an Acclaim™ PepMap™ 100 C18 HPLC column (3 μm particle size, 75 μm diameter, 150 mm length) and separate using an EASY-Spray™ PepMap™ Neo UHPLC column (2 μm C18 particle, 75 μm diameter,150 mm length) with LC conditions.
Buffer A: 0.1% formic acid
Buffer B: 80%ACN, 0.1% formic acid
Acquire data using MS settings.
| A | B | |
| Global Parameters | ||
| Application Mode | Peptide | |
| Method Duration (min) | 22.6 | |
| Settings | ||
| Infusion Mode | Liquid Chromatography | |
| Expected LC Peak Width (s) | 10 | |
| Advanced Peak Determination | ☑ | |
| Default Charge State | 2 | |
| Orbitrap Lock Mass Correction | Off | |
| Ion Source Properties | ||
| Ion Source Type | NSI | |
| Spray Voltage | Static | |
| Positive Ion (V) | 1900 | |
| Negative Ion (V) | 600 | |
| Ion Transfer Tube Temp (°C ) | 290 | |
| Use Ion Source Settings from Tune | ☐ | |
| FAIMS Mode | Not Installed | |
| Full Scan Properties | ||
| Orbitrap Resolution | 240000 | |
| Scan Range (m/z) | 380-980 | |
| RF Lens (%) | 40 | |
| AGC Target | Custom | |
| Normalised AGC Target (%) | 500 | |
| Maximum Injection Time (ms) | 5 | |
| Microscans | 1 | |
| Data Type | Profile | |
| Polarity | Positive | |
| Source Fragmentation | ☐ | |
| DIA MS Settings | ||
| Start Time (min) | 0 | |
| End Time (min) | 22.6 | |
| Precursor Mass Range (m/z) | 380-980 | |
| DIA Window Type | Auto | |
| Isolation Window (m/z) | 4 | |
| Window Overlap (m/z) | 0 | |
| Window Placement Optimisation | On | |
| Number of Scan Events | 149 | |
| DIA Window Mode | m/z Range | |
| Collision Energy Type | Normalised | |
| HCD Collision Energy (%) | 25 | |
| Detector Type | Astral | |
| TMT | Off | |
| Scan Range (m/z) | 150-2000 | |
| RF Lens (%) | 40 | |
| AGC Target | Custom | |
| Normalized AGC Target (%) | 500 | |
| Absolute AGC Value | 5.000e4 | |
| Maximum Injection Time (ms) | 3 | |
| Microscans | 1 | |
| Data Type | Centroid | |
| Polarity | Positive | |
| Source Fragmentation | Disabled | |
| Loop Control | Time | |
| Time (sec) | 0.6 |
Spectral Library Generation using DIA-NN Software (version 1.8.1)
Spectral Library Generation using DIA-NN Software (version 1.8.1)
Generate a predicted spectral library in DIA-NN by providing the downloaded reviewed (SwissProt) human UniProt FASTA file, searched with the following parameters:
Command
Use this newly created spectral library to search the 96 peptide fractions, enabling the generation of a new spectral library from the search output:
Command