title: Quantitative carbamylation as a stable isotopic labeling method for comparative proteomics.
abstract: A method was developed that uses urea to both solublize and isotopically label biological samples for comparative proteomics. This approach uses either light or heavy urea ((12)CH(4)(14)N(2)O or (13)CH(4)(15)N(2)O, respectively) at a concentration of 8 M and a pH of 7 to dissolve the samples prior to digestion. After the sample is digested using standard proteomic protocols and dried, isotopic labeling is completed by resuspending the sample in a solution of 8 M urea at a pH of 8.5, using the same isotopic species of urea as used for digestion and incubating for 4 h at 80 degrees C. Under these conditions, carbamylation occurs only on the primary amines of the peptides. The effects of complete carbamylation on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) (collision-induced dissociation (CID)) were examined. Peptides that had a C-terminal carbamylated lysine residue were found to have a reduced intensity when viewed by MALDI-TOFMS. CID of a tryptic peptide that was carbamylated on both the N-terminus and the C-terminus was found to have a more uniform distribution of b- and y-ions, as well as prominent ions from loss of water. Reversed-phase chromatography coupled to ESI-MS/MS was used to identify and quantify the isotopically labeled standard proteins, bovine serum albumin (BSA), bovine transferrin, and bovine alpha-casein. Quantitative error between theoretical and observed data ranged from 1.7-10.0%. Relative standard deviations for protein quantitation ranged from 5.2-27.8% over a dynamic range from 0.1-10 (L/H). The development of a method utilizing urea-assisted carbamylation of lysines and N-termini to globally labeled samples for comparative proteomics may prove useful for samples that require a strong chaotrope prior to proteolysis.
authors.affiliation: Complex Carbohydrate Research Center, The Departments of Biochemistry & Molecular Biology and Chemistry, University of Georgia, 315 Riverbend Road, Athens, GA 30302-4712, USA.
authors.forename: Peggi M
authors.initials: PM
authors.lastname: Angel
authors.forename: Ron
authors.initials: R
authors.lastname: Orlando
mesh_terms: D000818:Animals; D002138:Calibration; D002219:Carbamates / Q000138:chemical synthesis*; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D006868:Hydrolysis; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012015:Reference Standards; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014168:Transferrin / Q000737:chemistry; D014357:Trypsin
chemical_list: D002219:Carbamates; D002364:Caseins; D007202:Indicators and Reagents; D010446:Peptide Fragments; D014168:Transferrin; D012710:Serum Albumin, Bovine; D014357:Trypsin