Carbamylation of proteins in 2-D electrophoresis--myth or reality?
Carbamylation is widely quoted as being a problem in 2-D gel analysis and the associated sample preparation steps. This modification occurs when iso-cyanate, a urea break-down product, covalently modifies lysine residues, thus inducing a change in isoelectric point. Urea is used at up to 9 M concentrations in sample preparation and 2-D gels because of its ability to disrupt protein structure and effect denaturation without the need for ionic surfactants such as SDS. We have studied carbamylation using 7 M urea and 2 M thiourea, under a range of experimental temperatures to establish when, and if, it occurs and what can be done to minimize the modification. The actual time required for protein extraction from a tissue is usually short compared to the time required for procedures such as reduction and alkylation and IPG rehydration and focusing. Therefore, it is the temperature during these post-extraction procedures that is the most critical factor. Our experiments have shown that carbamylation does not occur during electrophoresis in the presence of urea, even with prolonged run-times. However, under poorly controlled sample preparation and storage conditions, it can become a major event.
McCarthy
John
J
Proteome Systems Limited, North Ryde, Sydney, NSW, Australia.
Hopwood
Femia
F
Oxley
David
D
Laver
Matthew
M
Castagna
Annalisa
A
Righetti
Pier Giorgio
PG
Williams
Keith
K
Herbert
Ben
B
D000478:Alkylation; D002219:Carbamates / Q000737:chemistry*; D005591:Chemical Fractionation / Q000379:methods; D003485:Cyanates / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002219:Carbamates; D003485:Cyanates; D011506:Proteins; C070766:carbamic acid; C005057:isocyanic acid