Oct 27, 2025

Public workspaceIn solution digestion (acetone precipitation)

DOI
https://dx.doi.org/10.17504/protocols.io.14egn6436l5d/v1
In solution digestion (acetone precipitation)
  • Cristina CARDENAL PERALTA1
  • 1University of Edinburgh
Cristina CARDENAL PERALTA
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn6436l5d/v1
Protocol Citation: Cristina CARDENAL PERALTA 2025. In solution digestion (acetone precipitation). protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6436l5d/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 19, 2024
Last Modified: October 27, 2025
Protocol Integer ID: 102103
Keywords: acetone precipitation, in solution digestion, proteomics strategy for comprehensive protein analysis, ms analysis for global proteome profiling, global proteome profiling, used proteomics strategy, comprehensive protein analysis, efficient enzymatic cleavage of protein, protein, enabling efficient enzymatic cleavage, protein complexity, solution digestion, peptide, acetone precipitation, digestion, facilitating downstream lc
Abstract
In-solution digestion is a widely used proteomics strategy for comprehensive protein analysis, enabling efficient enzymatic cleavage of proteins into peptides in a liquid-phase environment. This method preserves protein complexity while facilitating downstream LC-MS/MS analysis for global proteome profiling.
Guidelines
After acetone precipitation O/N, it might be difficult to resuspend the peptide pellet just by vortexing. Gentle pipetting is advised at this point to break apart the pellet more efficiently.
Materials
Buffers
TEAB (Triethylammonium bicarbonate)

Enzymes
Trypsin: ThermoFisher 90057 20 µg per vial

Reagents
200 mM STOCK: Dithiothreitol (DTT) or 200 mM STOCK TCEP (tris(2-carboxyethyl)phosphine)
500 mM STOCK: Iodoacetamide (IAA)
Acetone 100%

Troubleshooting
Safety warnings
IAA incubation needs to happen in the dark
Sample reduction and alkylation (DAY 1)
1h 10m
Resuspend 100 µg of protein per condition with 100 µL with Concentration100 millimolar (mM) TEAB.

Add 5 µL of the Concentration200 millimolar (mM) TCEP/DTT and incubate sample at Temperature55 °C for 40 min. Shake at 600-700 rpm.

40m
Add 5 µL Concentration500 millimolar (mM) IAA (making it a final concentration of Concentration25 millimolar (mM) ). Incubate at TemperatureRoom temperature in the dark for 30 min.

30m
Add six volumes (roughly 600 µL) of pre-chilled (Temperature-20 °C ) acetone and freeze at Temperature-20 °C . Allow precipitation O/N.

Overnight
Protein digestion (DAY 2)
10m
Centrifuge the samples at 8000 x g for 10 minutes.
10m
Resuspend 100 µg of acetone-precipitated protein pellets with 100 µL ofConcentration50 millimolar (mM) TEAB.
The pellet might not completely dissolve; however, after proteolysis at Temperature37 °C all the peptides will be solubilised.

Immediately before use, add 20 µL of the Trypsin Storage Solution to the bottom of the trypsin glass vial and incubate for 5 minutes.
Add 2.5 µL of trypsin per 100 µg of protein (initial protein). Digest sample O/N at Temperature37 °C

Overnight