title: Inhibition of protein carbamylation in urea solution using ammonium-containing buffers.
abstract: 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.
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Shisheng
authors.initials: S
authors.lastname: Sun
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Jian-Ying
authors.initials: JY
authors.lastname: Zhou
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
authors.forename: Weiming
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA. Electronic address: hzhang32@jhmi.edu.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D064751:Ammonium Compounds / Q000737:chemistry*; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000378:metabolism*; D002021:Buffers; D003485:Cyanates / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D012996:Solutions; D014357:Trypsin / Q000378:metabolism; D014508:Urea / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D064751:Ammonium Compounds; D001798:Blood Proteins; D002021:Buffers; D003485:Cyanates; D012996:Solutions; D014508:Urea; D014357:Trypsin; C005057:isocyanic acid
keywords: Ammonium-containing buffer; Carbamylation; Mass spectrometry; Proteomics; Urea