title: Protein carbamylation: in vivo modification or in vitro artefact?
abstract: 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.
authors: Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany.
authors: Laxmikanth
authors: L
authors: Kollipara
authors: René P
authors: RP
authors: Zahedi
mesh_terms: D000818:Animals; D016477:Artifacts; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D007782:Lactoglobulins / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D012710:Serum Albumin, Bovine / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D002364:Caseins; D006454:Hemoglobins; D007782:Lactoglobulins; D009211:Myoglobin; D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; D014508:Urea; D014357:Trypsin