Carbamylation (carbamoylation) of lysine residues and protein N-termini is a 
nonenzymatic PTM that has been related to protein ageing. In contrast to other 
PTM, such as phosphorylation, carbamylation can be artificially introduced 
during sample preparation with urea, thus affecting studies directed toward in 
vivo carbamylation. In aqueous solution, urea-commonly used for denaturing 
proteins-is in equilibrium with ammonium and isocyanate. Under alkaline 
conditions, the latter can react with primary amines of free N-termini and 
ε-amine groups of lysines to form carbamyl derivatives. Despite being a 
relatively slow process, which is accelerated at elevated temperatures, 
prolonged incubation of protein/peptide samples in urea buffers can induce 
undesired carbamylation, hampering not only the proteolytic digestion with 
trypsin and peptide identification by MS, but also interfering with stable 
isotope-labeling techniques such as iTRAQ, tandem mass tags, and isotope-coded 
protein labeling. Here, we evaluated the extent of urea-induced carbamylation 
under commonly used sample preparation conditions. From our results, we can 
deduce that carbamylation occurs in all cases involving urea, however with 
varying degree: e.g. carbamidomethylation in the presence of 8.0 M urea induced 
carbamylation of 17% of N-termini and 4% of Lys residues. Additionally, 
researching a recently published large-scale dataset revealed a high degree of 
urea-induced carbamylation in current proteomic samples.