Quantitative carbamylation as a stable isotopic labeling method for comparative proteomics

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°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. Copyright © 2007 John Wiley & Sons, Ltd.