In my experiment I perform the in-solution digestion in 6 M Urea and 2 M thiourea buffer (UTU, pH 8.0 with Tris-HCl) as follows: Reduction> alkylation> Lys-c digestion for 3 hours at room temperature > four-fold dilution of the UTU by 10 mM Tris-HCl (pH 8.0) > Overnight Trypsin digestion at room temperature > bring the pH around 2-3 by 0.2% TFA > Speed vac the peptide to dryness and resolve with 1 M glycolic acid, 5%TFA, and 80% ACN.
Now, during the speed vac, I am using high temperature, will this carbamylate the peptides? As far as I know, the carbamylation takes place during the digestion.
The high-temperature speed vac accelerates the drying and concentrating process.
Urea along with the protein and heat is an ultimate invitation for carbamylation. The issue with the urea is that even if it is pure it will degrade to isocyanic acid. Can you use GdnHcl instead?
Hi, Mahesh, Thanks for your suggestion.
We thought to use GdnHCl instead of Urea-Thiourea (UTU). However, we found in litterateurs that UTU works better in case of membrane protein denaturation. I am thinking to prepare the UTU with 1M NH4HCO3 instead of Tris-HCl. There are some studies showed that NH4HCO3 works better in scavenging cyanate during the digestion.
My main query was, how much can it affect after the digestion, which is the speed vac dryness in high temperature.
You can evaporate at room temperature and can also use the filter-aided sample preparation (FASP) method. 1M NH4HCO3 is an alternative method. Several people use speed vac, and how much it affects really depends on your protein of interest and temp. If you want to know with your system then you can simply take speedvac sample and sample dried via other method say freeze dry - send them for analysis then from that result you can calculate carbamylation ratio using peak area of C peptides / Peak area of non- C peptides.
Thanks for the alternative Ideas. This time I prepared the samples using with 10 mM Tris-HCl and 1M NH4HCO3 and speed vac'd at low heat (which is around 40-43 C in the Tomy Speed vac) and running in our Orbitrap XL. I can get the data soon and let you know the carbamylation ratio. Thanks again.
The low heat speed vac worked fine just took a little bit longer than the high temp. The trade off would be worthy if the Carbamylation rate goes down.
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