title: Enhancing the stability of ¹⁸O-labeled peptides through removal of immobilized trypsin by ZipTips.
abstract: Trypsin-catalyzed ¹⁸O labeling is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by the instability of ¹⁸O-labeled peptides caused mainly by oxygen back-exchange. Although a number of attempts have been made to reduce or prevent oxygen back-exchange, there is still room for improvement. Here we demonstrate that the removal of immobilized trypsin by filtration using ZipTips can efficiently minimize oxygen back-exchange and enhance the stability of ¹⁸O-labeled peptides under various pH conditions. The ¹⁸O-labeled peptides processed by the approach were successfully separated by immobilized pH gradient-isoelectric focusing (IPG-IEF), and no marked decrease in the extent of labeling was observed. The results also demonstrated that there was no correlation between the extent of ¹⁸O labeling and molecular weight or isoelectric point (pI). The approach presented here is especially applicable to microscale samples. Its ability to generate stably ¹⁸O-labeled samples without back-exchange should expand the application scope of the ¹⁸O-labeling technique.
authors.affiliation: Xiangya Hospital, Central South University, Changsha, China.
authors.forename: Mao-Yu
authors.initials: MY
authors.lastname: Li
authors.forename: Fang
authors.initials: F
authors.lastname: Peng
authors.forename: Jian-Hong
authors.initials: JH
authors.lastname: Zuo
authors.forename: Hong
authors.initials: H
authors.lastname: Yi
authors.forename: Can-E
authors.initials: CE
authors.lastname: Tang
authors.forename: Cui
authors.initials: C
authors.lastname: Li
authors.forename: Peng-Fei
authors.initials: PF
authors.lastname: Zhang
authors.forename: Zhu-Chu
authors.initials: ZC
authors.lastname: Chen
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Xiao
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005374:Filtration / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D007525:Isoelectric Focusing / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D010103:Oxygen Isotopes / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D055550:Protein Stability; D040901:Proteomics / Q000379:methods; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D055767:Immobilized Proteins; D010103:Oxygen Isotopes; D010455:Peptides; D012710:Serum Albumin, Bovine; D014357:Trypsin