Optimization and quality assessment of the post-digestion O labeling based on urea for protein denaturation by HPLC/ESI-TOF mass spectrometry

The post-digestion ¹⁸O labeling method decouples protein digestion and peptide labeling. This method allows labeling conditions to be optimized separately and increases labeling efficiency. A common method for protein denaturation in proteomics is the use of urea. Though some previous studies have used urea-based protein denaturation before post-digestion ¹⁸O labeling, the optimal ¹⁸O labeling conditions in this case have not been yet reported. Present study investigated the effects of urea concentration and pH on the labeling efficiency and obtained an optimized protocol. It was demonstrated that urea inhibited ¹⁸O incorporation depending on concentration. However, a urea concentration between 1 and 2 M had minimal effects on labeling. It was also demonstrated that the use of FA to quench the digestion reaction severely affected the labeling efficiency. This study revealed the reason why previous studies gave different optimal pH for labeling. They neglect the effects of different digestion conditions on the labeling conditions. Excellent labeling quality was obtained at the optimized conditions using urea 1–2 M and pH 4.5, 98.4 ± 1.9% for a standard protein mixture and 97.2 ± 6.2% for a complex biological sample. For a 1:1 mixture analysis of the ¹⁶O- and ¹⁸O-labeled peptides from the same protein sample, the average abundance ratios reached 1.05 ± 0.31, demonstrating a good quantitation quality at the optimized conditions. This work will benefit other researchers who pair urea-based protein denaturation with a post-digestion ¹⁸O labeling method.