Jan 01, 2026
In-Gel Trypsin digestion of Proteins for MS analysis
- Catarina Correia1,2,
- Maria da Conceição Almeida1,2,
- Ana Catarina Guerreiro1,2,
- Ricardo Gomes1,2,
- Ana Luísa Simplício1,2,
- Patrícia Gomes-Alves1,2,
- Isabel A. Abreu1,2
- 1Instituto de Tecnologia Química e Biológica, Universidade NOVA de Lisboa (ITQB NOVA);
- 2Instituto de Biologia Experimental e Tecnológica (iBET)
- Catarina Correia: Wrote and validated the Protocol;
- Maria da Conceição Almeida: Validated the protocol
- Isabel A. Abreu: Revised the published protocol
Protocol Citation: Catarina Correia, Maria da Conceição Almeida, Ana Catarina Guerreiro, Ricardo Gomes, Ana Luísa Simplício, Patrícia Gomes-Alves, Isabel A. Abreu 2026. In-Gel Trypsin digestion of Proteins for MS analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvje9mbgk5/v1
Manuscript citation:
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 02, 2025
Last Modified: January 01, 2026
Protocol Integer ID: 226239
Keywords: Proteome ID, In-gel digestion, Trypsin, Peptide cleanup, Mass spectrometry, Proteomics workflow, Sample preparation, gel trypsin digestion of protein sample, gel trypsin digestion of protein, proteomic identification via mass spectrometry, gel trypsin digestion, protein sample, proteomic identification, mass spectrometry, quality peptide, protein, ms analysis this protocol, ms analysis
Abstract
This protocol describes a standardized workflow for in-gel trypsin digestion of protein samples intended for proteomic identification via mass spectrometry (MS). This procedure ensures the production of high-quality peptides while minimizing contamination and ensuring reproducibility and efficiency.
Guidelines
- Always handle samples under clean, keratin‑controlled conditions. Wear powder‑free gloves (preferably nitrile), a lab coat and a hair cover at all times to minimize the risk of keratin contamination.
- Use mass-spectrometry-compatible staining methods such as Coomassie Blue (see Material tab). Do not use fixatives such as glutaraldehyde or paraformaldehyde.
- Preferably excise bands under a laminar‑flow hood or under water to minimize keratin contamination and on a clean glass plate to avoid plastic‑derived contaminants. Clean all tools and the cutting surface with 70% ethanol between each band to prevent cross‑contamination.
- Keep gel pieces fully submerged at all times; increase the solution volume as needed to maintain immersion and allow free movement during shaking.
- Use LC-MS grade reagents.
- Use low-protein binding microtubes (avoid autoclaved or PCR-coated microtubes) and low-binding pipette tips.
Materials
- Ice bucket
- Lab coat and hair cover
- Low-binding pipette tips
- Low‑protein‑binding microtubes (1.5–2 mL)
- Powder‑free gloves (preferably nitrile)
- Stainless steel scalpel blade and handle
Kits, Reagents & Solutions
- 70% ethanol
- Acetonitrile (ACN) LC-MS grade
- Ammonium bicarbonate (NH₄HCO₃) – 50 mM in Water and in 50% ACN
- C18 ZipTips (e.g.: OMIX C18 tips ref.: HPAA5700310)
- Dithiothreitol (DTT) – 10 mM in 50 mM NH₄HCO₃
- Formic acid (FA) LC‑MS grade
- Hydrochloric acid (HCl) – 1 mM
- Ice
- Iodoacetamide (IAA) – 55 mM in 50 mM NH₄HCO₃
- Mass-spectrometry-compatible stain
- PAGE Gel Preparation Kit
- Trypsin (e.g., Promega V5111) – 0.1 µg/µL in 1 mM HCl
- Water LC‑MS grade
Mass-spectrometry-compatible stains
- CoomassieTM Brilliant Blue G-250
- CoomassieTM Brilliant Blue R-250
- ImperialTM Protein Stain (Thermo Scientific)
- PageBlueTM Protein Staining Solution (Thermo Scientific)
- PierceTM Silver Stain Kit (Thermo Scientific)
- SimplyBlueTM SafeStain (Invitrogen)
Alternative Proteases
- Asp‑N (e.g., Roche 11420488001)
- Chymotrypsin (e.g., Roche 11418467001)
- Glu‑C (e.g., Roche 11047817001)
- Trypsin + Lys‑C (e.g., Promega PROMV5072)
Equipment
- Electrophoresis chamber system and power supply
- Fridge and freezer
- Platform rocker
- SpeedVac concentrator
- Spin-down centrifuge
- Thermomixer (capable of 37°C, 56°C, 70°C and 650 rpm agitation)
- Ultrasonic bath
- Variable volume single channel pipettes
- Vortex mixer
Troubleshooting
Safety warnings
- Be careful not to aspirate the dried gel pieces into the pipette tip as the gel pieces shrink after dehydration and they also can stick to the pipette tip.
- Incubate Iodoacetamide (IAA) step in the dark to avoid side reactions (step 10).
- Handle Formic acid (FA) with the appropriate personal protective equipment (PPE) and follow the corresponding waste‑management procedures.
- Store Trypsin stock aliquots at -20 °C for up to 6 months and avoid repeated freeze-thaw cycles.
Protein electrophoresis separation
4h 15m
Run the protein samples on an SDS–PAGE gel and stain the gel using a mass-spectrometry-compatible stain such as Coomassie Blue (see Material tab).
Note: To minimize keratin contamination, perform all steps of this protocol under clean, keratin‑controlled conditions. Wear powder‑free gloves (preferably nitrile), a lab coat and a hair cover at all times.
2h
Destain the gel as thoroughly as possible, as residual dye can interfere with mass spectrometry data acquisition.
2h
Excise the protein bands of interest from the gel and cut them into 1–2 mm³ pieces to improve solvent exchange. Excise only the well‑defined, darkest region, prioritizing protein concentration over total band area to increase target purity and improve in‑gel digestion efficiency.
Note: Preferably excise bands under a laminar‑flow hood or under water to minimize keratin contamination and on a clean glass plate to avoid plastic‑derived contaminants. Clean all tools and the cutting surface with 70% ethanol between each band to prevent cross‑contamination.
15m
Transfer the gel pieces into a low-protein binding microtube and spin down to bring them to the bottom of the tube.
Destaining coomassie stained protein band(s)
1h
Wash the gel pieces with water for 15 min with gentle agitation.
Remove and discard the supernatant.
15m
Add 50 mM ammonium bicarbonate (NH₄HCO₃) prepared in 50% acetonitrile (ACN) and incubate for 15 min with gentle agitation.
Remove and discard the supernatant.
15m
Repeat the above steps until the gel pieces are visibly clear (colorless). If needed, heat to 37 °C to help destaining, as residual dye can hinder protease access and peptide recovery.
30m
Protein reduction and alkylation
1h 45m
Add ACN, vortex and incubate for 15 min.
Spin down, remove and discard the supernatant.
Note: After dehydration, the gel pieces shrink and may adhere to the pipette tip. Take care not to aspirate them accidentally or transfer them to another microtube.
15m
Add 10 mM dithiothreitol (DTT) prepared in 50 mM NH4HCO3 and incubate for 45 min at 56ºC with agitation (650 rpm) to reduce disulfide bridges.
Spin down, remove and discard the supernatant.
45m
Add ACN, vortex and incubate for 15 min.
Spin down, remove and discard the supernatant.
15m
Add 55 mM iodoacetamide (IAA) prepared in 50 mM NH4HCO3 and incubate for 30 min at room temperature, protected from light, to alkylate the reduced disulfide bridges.
Remove and discard the supernatant.
30m
In-gel protein digestion
18h 10m
Add ACN, vortex and incubate for 15 min. Remove and discard the supernatant.
Repeat until the gel pieces are fully dehydrated (typically appearing white, shrunken and rigid).
30m
Vortex to separate the gel pieces and spread any residual droplets along the tube walls, then allow them to dry for 10 min at 37 °C with the lid open and without agitation.
Repeat until the gel pieces are fully dry.
30m
Reconstitute the contents of one 20 µg vial of trypsin (e.g., Promega V5111) in 200 µL of 1 mM HCl. Aliquot the reconstituted trypsin into 10 µL portions and store at −20 °C for up to 6 months.
10m
Place the required aliquots on ice and dilute each with 90 µL of 50 mM NH₄HCO₃ to obtain a 10 ng/µL working solution.
Note: Keep the working solution on ice, use immediately and avoid refreezing.
Add the trypsin working solution to the gel pieces and incubate on ice for 60 minutes.
Note: Check the samples every 15 min. If the initial volume has been fully absorbed by the gel pieces, add additional 50 mM NH₄HCO₃ (without enzyme) to ensure the gel pieces remain completely covered.
1h
Remove any excess buffer and add fresh 50 mM NH₄HCO₃ to fully cover the gel pieces and allow them to move during shaking. Incubate overnight at 37 °C with agitation (650 rpm).
Alternative Proteases and Conditions:
- Trypsin+Lys-C or Chymotrypsin
Concentration: 10 ng/µL
Digestion buffer: 50 mM NH₄HCO₃
Temperature: 37ºC
- Asp-N
Concentration: 20 ng/µL
Digestion buffer: 50 mM NH₄HCO₃
Temperature: 37ºC
- Glu-C
Concentration: 25 ng/µL
Digestion buffer: 25 mM NH₄HCO₃
Temperature: 25ºC
16h
Spin down and transfer the supernatant (containing the digested peptides) to a new low-protein binding microtube.
Stop the digestion by acidifying the digested sample with Formic acid (FA) to a final concentration of 5%.
Peptides extraction with ACN (if necessary)
1h 55m
Dehydrate the gel pieces with ACN for 10 min in an ultrasonic bath.
Transfer the ACN extract to the microtube containing the digested sample.
10m
Rehydrate the gel pieces with water for 10 min in an ultrasonic bath.
Transfer the remaining water to the same microtube containing the digested sample.
10m
Repeat the last two steps.
20m
Dry the combined extract completely using a SpeedVac.
1h
Resuspend the digested sample in 100 µL of 5% FA by vortexing for 5 min, then sonicate for 10 min to ensure complete peptide solubilization.
15m
Peptides purification with C18 filtered pipet tips
1h 5m
Aspirate 100 µL of 50% ACN to clean/activate the C18 tip.
Discard the solution and repeat once more.
1m
Aspirate 100 µL of 5% FA to equilibrate the C18 tip.
Discard the liquid and repeat once more.
1m
Aspirate and dispense the sample five times to ensure maximum peptide binding to the C18 tip.
1m
Aspirate 100 µL of 5% FA to clean the peptides.
Discard the supernatant and repeat once more.
1m
Aspirate 100 µL of 90% ACN + 0.1% FA and elute the purified peptides into a new low-protein binding microtube.
1m
Dry the purified peptides completely using a SpeedVac.
1h
Store the purified samples, the flow‑throughs and the remaining gel pieces at −20 °C.
Contact Information
Mass Spectrometry Unit (UniMS)
E-mail: [email protected]
Site: http://www.itqb.unl.pt/facilities/UniMS/
Acknowledgements
Mass Spectrometry Unit
Av. República, Oeiras, Portugal
Tel. 351 – 214469451/52; http://www.itqb.unl.pt/facilities/UniMS/