Oct 24, 2025
On-bead digestion under denaturing conditions
- 1University of Edinburgh
Protocol Citation: Cristina CARDENAL PERALTA 2025. On-bead digestion under denaturing conditions. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32kypv25/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: June 24, 2024
Last Modified: October 24, 2025
Protocol Integer ID: 102292
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
Abstract
On-bead digestion is used for mass spectrometry to streamline the sample preparation process, reducing sample loss and contamination risks. It allows for efficient protein digestion directly on the solid support, enhancing peptide recovery and improving the sensitivity and accuracy of mass spectrometry analysis by minimizing handling steps and ensuring more consistent results.
Guidelines
Perform all steps at Room temperature .
Materials
Buffers:
Denaturation buffer: 8M urea in 50 mM ABC. (For membrane proteins use 7M urea/2M thiourea).
Digestion buffer: 50 mM ABC (ammonium bicarbonate) in water, pH 8.
Reagents:
DTT: 1 M in 50 mM ABC.
IAA: 1M in 50 mM ABC
Enzymes:
LysC: 1 µg/ µL in 0.1% TFA
Trypsin: 1 µg/ µL in 0.1% TFA
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
When the samples arrive isolate beads from the purification buffer using a magnetic tube rack. Allow 5 minutes of incubation and remove purification buffer.
5m
Resuspend the beads in three times the original bead volume using denaturation buffer
Reduction/Alkylation
1h
Add 1 µL of 1 Molarity (M) DTT per 10 µL of sample volume and incubate for 30 min atRoom temperature . Shake at 1200 rpm.
30m
Add 1 µL of 1 Molarity (M) IAA per 10 µL of sample volume and incubate for 30 min atRoom temperature . Shake at 1200 rpm in the dark.
30m
Digestion
4h
Add 1 µg of LysC solution per 50 µg of protein and incubate for at least 3-4 hours at Room temperature .
4h
Dilute sample with 5x digestion buffer.
Add 4 µg of trypsin per 50 µg of protein and incubate O/N at 37 °C .
The trypsin and the LysC digestion steps are interchangeable, however, trypsin does not work in high urea concentrations, and dilution of the sample is needed before adding trypsin.
Sample acidification
Separate beds from supernatant by using a magnetic tube rack (allow 5 min for all the beads to stick to the side of the tube). Collect supernatant into a new Eppendorf tube.
Stop the digestion by acidifying the sample to pH < 2.5 with 10 % TFA, to a final % of 1%.
Proceed to load the sample onto equilibrated C18 StageTips (samples can stay in the fridge until the tips are equilibrated).