Enhancing the stability of ¹⁸O-labeled peptides through removal of immobilized trypsin by ZipTips.
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.
Li
Mao-Yu
MY
Xiangya Hospital, Central South University, Changsha, China.
Peng
Fang
F
Zuo
Jian-Hong
JH
Yi
Hong
H
Tang
Can-E
CE
Li
Cui
C
Zhang
Peng-Fei
PF
Chen
Zhu-Chu
ZC
Xiao
Zhi-Qiang
ZQ
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*
D055767:Immobilized Proteins; D010103:Oxygen Isotopes; D010455:Peptides; D012710:Serum Albumin, Bovine; D014357:Trypsin