Propionylation and tryptic digestion
Sigrid Verhelst, Laura Corveleyn
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
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).
Steps
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
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.
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
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
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
