Does anyone know if 5 M Guanidine will artifactually modify amino acids in your peptide sequence. 8 M urea will carbamylate your peptide, but what about guanidine?
Dear Norelle,
Attached please find a review article entitled "Analysis of protein carbonylation — pitfalls and promise in commonly used methods " published in Free Radical Research, October 2014; 48(10): 1145–1162 which indicates that indeed 5M guanidine does induce artifactual PTMs; please see Table 1 in page 14, last row (concentrations of 3-6 M can do the job).
Hoping this will be helpful,
Rafik
Thanks Rafik, but I am interested in Carbamylation not Carbonylation.
Dear Norelle,
Attached is a paper discusses the Carbamylation.
The following is the paper experimental section:
EXPERIMENTAL PROCEDURES
Isolation and Carbamylation of Lens Crystallins-Bovine aA-crystallins
were isolated and identified as previously reported (Smith et
al., 1991). Homogenates of whole bovine lenses were separated by gel
filtration into the a-, p-, and y-crystallin fractions using Sephadex
G-200. The a-crystallins (500 pg/ml) were carbamylated by incubation
in 0.1 M KNCO, 0.2 M NHIOAc containing 0.02% NaN3 at pH7.2 and 37 "C for various times. Complete (or nearly complete)
modification was effected by incubating the protein (300 pg/ml) in
the same buffer, but with 6 M guanidine hydrochloride, at 37 "C for
24 h. A control was prepared by incubating a-crystallins in the same
buffer with 0.1 M KC1 in place of the 0.1 M KNCO. After incubation,
the samples were dialyzed against water to remove salts. The acidic
(aA) and basic (aB) forms of the a-crystallins were separated by
reversed-phase HPLC (Rainin Instrument Co., Woburn, MA) using
a Vydac 0.46 X 25-cm CI8 column with UV detection at 280 nm. A
linear gradient of 30-60% CH3CN in HzO, both with 0.1% of trifluoroacetic
acid, was used to elute the proteins. The gene products, aA2
and aB2, coelute with their respective phosphorylated forms aAl and
aB1 and aBO (Smith et aZ., 1991, 1992). The fractions corresponding
to aA-crystallin were collected, freeze-dried, and stored at -10 "C
until analyzed.
Hoping this will help,
Rafik
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