Ultrafiltration to remove trypsin for suppressing the back-exchange of ¹⁸O labeling

The post-digestion of ¹⁸O labeling is widely used in comparative proteomics for the relative quantification of proteins and peptides. Application and precise quantitative analysis are hindered, since ¹⁸O labeling is pH sensitive and has back-exchange. Herein, in a study of peptides derived from BSA (bovine serum albumin), we demonstrated through a detailed evaluation that removal of soluble trypsin by ultrafiltration prevented back-exchange and effectively enhanced the stability of ¹⁸O-labeled peptides under off-gel separation even without trypsin inhibitor. After ultrafiltration, the ¹⁸O labeling efficiency was 95.8 ± 2.3% under off-gel separation. In addition, these peptides had a relative high, approximately 80% recovery and less than 5% ¹⁶O/¹⁸O ratio variation through ultrafiltration, indicating no apparent effect on quantification. Hence, the useful and economical method presented here effectively inhibited back-exchange in off-gel separation and might enable further applications towards large-scale biomarker discovery.