May 21, 2026
Version 2
In solution digestion DRAFT V.2
Version 1 is forked from On-bead digestion under denaturing conditions
This protocol is a draft, published without a DOI.
- 1University of Edinburgh
Protocol Citation: Cristina CARDENAL PERALTA 2026. In solution digestion DRAFT. protocols.io https://dx.doi.org/Version created by Amber Minhas
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: May 19, 2026
Last Modified: May 21, 2026
Protocol Integer ID: 317477
Keywords: beads, denaturing conditions, urea, trypsin, bead digestion, mass spectrometry, mass spectrometry analysis, efficient protein digestion, accuracy of mass spectrometry analysis, peptide recovery, enhancing peptide recovery, sample preparation process, solution digestion draft, solution digestion
Abstract
In solution digestion draft
Guidelines
Perform all steps at Room temperature .
Prepare denaturation buffer freshly on the day of the experiment.
Materials
Buffers:
Denaturation buffer: 8M urea in 50 mM ABC (ammonium bicarbonate)
Digestion buffer: 50 mM ABC in water, pH 8.
Reagents:
DTT: 1 M in 50 mM ABC.
IAA: 1M in 50 mM ABC
Formic acid
Enzymes:
Lys-C: 1 µg/µL stock solution
Trypsin: 1 µg/µL stock solution
Safety warnings
In this procedure all steps are done at Room temperature to reduce unwanted denaturization of amino acid side-chains by denaturing agents. Never heat your sample.
Sample reception
5m
Thaw cell pellet on ice
5m
Add 500 µL denaturation buffer to the cell pellet and resuspend thoroughly.
If the pellet is not fully solubilised or remains highly viscous, increase the volume up to 1 mL total.
Lysis and protein extraction
Disrupt the cells with vigorous pipetting
Centrifuge lysate at 16,000 rcf, 10 min, 4°C to pellet insoluble debris and transfer the supernatant to a new tube.
Determine protein concentration using the BCA assay. Prepare 1:10 and 1:20 lysate dilutions and use the dilution that falls within the reliable range of the BSA standard curve. Calculate the volume of lysate required for 30 µg protein input.
Reduction/Alkylation
1h
Make a working solution of DTT (100mM from 1M)
Add DTT to a final concentration of 10 mM and incubate for 30 min at 22 °C. Shake at 1200 rpm.
30m
Make a working solution of IAA (100mM from 1M)
Add IAA to a final concentration of 20 mM and incubate for 30 minutes at 22 °C in the dark. Shake at 1200 rpm.
30m
Digestion
4h
No Lys-C added for initial optimisation. Incubate sample for 4 h at 22 °C with shaking at 1200 rpm
4h
Centrifuge at high speed to clarify the lysate (5,000 × g, 22 °C, 5 min) and transfer the supernatant to a new tube
Digestion
4h
Dilute the sample by adding 4x volumes of digestion buffer to reduce urea concentration prior to trypsin digestion.
Digestion
4h
Resuspend trypsin vial in 20 µL 0.1% TFA and 80 µL ABC to generate a 0.2 µg/µL trypsin solution. Add 5 µL trypsin solution (~1 µg trypsin) per sample. Incubate overnight at 37°C.
Incubate overnight at 37 °C
Sample acidification
Add formic acid to a final concentration of 1% to quench digestion
Centrifuge briefly to pellet insoluble material and transfer the peptide-containing supernatant to a new tube
Proceed to load the sample onto equilibrated C18 StageTips (samples can stay in the fridge until the tips are equilibrated)