Jan 01, 2026
In-Solution 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-Solution Trypsin digestion of Proteins for MS analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7n919lwz/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: October 16, 2025
Last Modified: January 01, 2026
Protocol Integer ID: 230007
Keywords: Proteome ID, In-solution digestion, Trypsin, Peptide cleanup, Mass spectrometry, Proteomics workflow, Sample preparation, solucion trypsin digestion of protein sample, proteomic identification via mass spectrometry, solution trypsin digestion of protein, solucion trypsin digestion, protein sample, solution trypsin digestion, proteomic identification, mass spectrometry, quality peptide, protein, ms analysis this protocol, ms analysis
Abstract
This protocol describes a standardized workflow for in-solucion 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.
- Ideally, the sample should have a protein concentration of 1 mg/mL.
- Precipitate samples to remove contaminants incompatible with trypsin digestion, including protease inhibitors, detergents/surfactants (e.g., SDS, Triton X‑100), chaotropes (urea, guanidine), chelators (EDTA/EGTA), organic solvents, salts/buffers (e.g., Tris, phosphate, HEPES), glycerol and unquenched alkylating agents.
- Use LC-MS grade reagents.
- Use low-protein binding microtubes (avoid autoclaved or PCR-coated microtubes) and low-binding pipette tips.
Materials
- Lab coat and hair cover
- Low-binding pipette tips
- Low‑protein‑binding microtubes (1.5–2 mL)
- Powder‑free gloves (preferably nitrile)
Kits, Reagents & Solutions
- Acetone HPLC grade
- Ammonium bicarbonate (NH₄HCO₃) – 50 mM or 1 M in Water
- C18 ZipTips (e.g.: OMIX C18 tips ref.: HPAA5700310)
- Dithiothreitol (DTT) – 250 mM in water
- Formic acid (FA) LC‑MS grade
- Hydrochloric acid (HCl) – 1 mM
- Iodoacetamide (IAA) – 500 mM in water
- Protein Quantification Kit
- Trypsin (e.g., Promega V5111), 0.1 µg/µL in 1 mM HCl
- Water LC‑MS grade
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
- Centrifuge with 1.5-2 ml microtubes rotor
- Fridge and freezer
- SpeedVac concentrator
- Spin-down centrifuge
- Thermomixer (capable of 37°C, 56°C and 650 rpm agitation)
- Ultrasonic bath
- Variable volume single channel pipettes
- Vortex mixer
Troubleshooting
Safety warnings
- Incubate Iodoacetamide (IAA) step in the dark to avoid side reactions (step 3).
- 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 reduction and alkylation
1h 50m
Quantify the sample before beginning the protocol. Ideally, the sample should have a protein concentration of 1 mg/mL.
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.
30m
Add 250 mM dithiothreitol (DTT) to the sample to reach a final concentration of 10 mM and incubate for 40 min at 56 °C, with agitation (650 rpm), to reduce disulfide bridges.
Allow the sample to cool afterwards.
40m
Add 500 mM iodoacetamide (IAA) to the sample to reach a final concentration of 20 mM and incubate for 30 min at room temperature, protected from light, to alkylate the reduced disulfide bridges.
30m
Add 250 mM DTT to the sample to raise the final DTT concentration to 21 mM and incubate for 10 min at room temperature, protected from light, to quench any remaining IAA.
10m
Protein precipitation (if necessary)
16h 30m
Precipitate the sample by adding six volumes of ice-cold acetone, vortex briefly to mix and incubate overnight at −20°C.
16h
Centrifuge at 16,000 × g for 10 minutes using a pre-cooled rotor. Carefully remove the supernatant and allow the pellet to air-dry for 5 min.
15m
Resuspend the pellet in 50 mM ammonium bicarbonate (NH₄HCO₃) by vortexing for 5 min, then sonicate for 10 min to ensure complete protein solubilization.
15m
In-sol protein digestion
16h 10m
Add 1 M NH₄HCO₃ to non‑precipitated samples to reach a final concentration of 50 mM NH₄HCO₃.
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
Add trypsin to the sample at a 1:50 (enzyme:protein, w/w) ratio and 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 stop the digestion by acidifying with Formic acid (FA) to a final concentration of 5%.
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 and the flow‑throughs 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/