title: Optimization and quality assessment of the post-digestion 18O labeling based on urea for protein denaturation by HPLC/ESI-TOF mass spectrometry.
abstract: The post-digestion (18)O 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 (18)O labeling, the optimal (18)O 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 (18)O incorporation depending on concentration. However, a urea concentration between 1 and 2M 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 (16)O- and (18)O-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 (18)O labeling method.
authors.affiliation: School of Life Science, Beijing Institute of Technology, Beijing 100081, China.
authors.forename: Hongbin
authors.initials: H
authors.lastname: Wang
authors.forename: Gaofei
authors.initials: G
authors.lastname: Hu
authors.forename: Yongqian
authors.initials: Y
authors.lastname: Zhang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Yuan
authors.forename: Xuan
authors.initials: X
authors.lastname: Zhao
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhu
authors.forename: De
authors.initials: D
authors.lastname: Cai
authors.forename: Yujuan
authors.initials: Y
authors.lastname: Li
authors.forename: Shengyuan
authors.initials: S
authors.lastname: Xiao
authors.forename: Yulin
authors.initials: Y
authors.lastname: Deng
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006868:Hydrolysis; D007553:Isotope Labeling / Q000379:methods*; D010103:Oxygen Isotopes / Q000737:chemistry*; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D014508:Urea / Q000737:chemistry*
chemical_list: D010103:Oxygen Isotopes; D011506:Proteins; D014508:Urea