Urea solution is one of the most commonly employed protein denaturants for 
protease digestion in proteomic studies. However, it has long been recognized 
that urea solution can cause carbamylation at the N termini of proteins/peptides 
and at the side chain amino groups of lysine and arginine residues. 
Protein/peptide carbamylation blocks protease digestion and affects protein 
identification and quantification in mass spectrometry analysis by blocking 
peptide amino groups from isotopic/isobaric labeling and changing peptide charge 
states, retention times, and masses. In addition, protein carbamylation during 
sample preparation makes it difficult to study in vivo protein carbamylation. In 
this study, we compared the peptide carbamylation in urea solutions of different 
buffers and found that ammonium-containing buffers were the most effective 
buffers to inhibit protein carbamylation in urea solution. The possible 
mechanism of carbamylation inhibition by ammonium-containing buffers is 
discussed, and a revised procedure for the protease digestion of proteins in 
urea and ammonium-containing buffers was developed to facilitate its application 
in proteomic research.