Jan 31, 2024
Propionylation and tryptic digestion
- 1ProGenTomics, Laboratory of Pharmaceutical Biotechnology, Ghent University, Ghent, Belgium
Protocol Citation: Laura Corveleyn, Sigrid Verhelst 2024. Propionylation and tryptic digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pz36g2w/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: January 31, 2024
Last Modified: January 31, 2024
Protocol Integer ID: 94452
Funders Acknowledgement:
IWT‐Vlaanderen
Grant ID: SB‐11179
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Abstract
Protocol to derivatize (propionylation) and digest histone proteins into peptides with the purpose of bottom-up label free LC-MS/MS analysis. In this method, aspecific overpropionylation at serine (S), threonine (T) and tyrosine (Y) is reversed by adding hydroxylamine (HA).
First propionylation
First propionylation
Start with vacuum dried sample (20 µg/sample)
Add 20 µL 1M triethylammonium bicarbonate (TEAB)
Add 20 µL prop-reagent (Isopropylalcohol:propionic anhydride (79:1))
Spin down & Incubate at room temperature for 30 minutes
Add 20 µL H2O
Spin down & Incubate at 37°C for 30 minutes
Vacuum dry sample
Trypsin digest
Trypsin digest
Calculate the amount of 500 mM TEAB, Calciumchloride (CaCl2), acetonitrile (ACN) and trypsin necessary to result in a 1:20 ratio (w/w) (1 µg trypsin/20 µg histones) in a final volume of 50µL.
Note
Final conc CaCl2: 1mM
Final conc ACN: 5%
Add correct volume of 500 mM TEAB, CaCl2 and ACN
Resuspend trypsin in 500 mM TEAB
Add trypsin at a 1:20 ratio (w/w) => 1 µg trypsin/20 µg histones
Spin down & incubate overnight at 37°C
Vacuum dry sample
Second propionylation
Second propionylation
Vacuum dried sample (20 µg/sample)
Add 20 µL 1M TEAB
Add 20 µL Prop-reagent (Isopropylalcohol:propionic anhydride (79:1))
Spin down & Incubate at room temperature for 30 minutes
Add 20 µL H2O
Spin down & Incubate at 37°C for 30 minutes
Vacuum dry sample
Reversing overpropionylation hydroxylamine mediated
Reversing overpropionylation hydroxylamine mediated
Vacuum dried sample (20 µg/sample)
Add 50 µL 0.5 M hydroxylamine (NH2OH)
Add 15 µL ammonium hydroxide (NH4OH) at pH 12
Spin down & Incubate at room temperature for 20 minutes
Adjust pH with formic acid (FA): 30 µl 100% FA
Vacuum dry sample
Note
Store in -20°c or -80°C until further use
Protocol references
Meert P, Dierickx S, Govaert E, De Clerck L, Willems S, Dhaenens M, Deforce D. Tackling aspecific side reactions during histone propionylation: The promise of reversing overpropionylation. Proteomics. 2016 Jul;16(14):1970-4. doi: 10.1002/pmic.201600045. PMID: 27139031; PMCID: PMC5096241.