Abstract
Small peptides have attracted increasing attention for their unique features and diverse biological func-tions.Achieving rapid separation and accurate quantification,however,remains a challenge because of their low abundance and the co-existence of numerous structural isomers.In this study,we developed a novel approach using isotope chemical labeling for ultrasensitive determination of di/tripeptides in bi-ological samples.We successfully synthesized a novel derivatization reagent,4-(2-(ethoxymethylene)-3-oxobutanamido)-N,N,N-trimethylbenzenaminium iodide(EOTMBA)as well as its deuterium-labeled iso-tope reagent(d3-EOTMBA).A total of 97 small peptides,including 89 dipeptides and 8 tripeptides,could be completely derivatized in methanol within 1.5 h at 60 ℃.After EOTMBA labeling,analysis of these di/tripeptides were achieved within 22 min by LC-MS/MS analysis.The method demonstrated 86.3％-113％accuracy and the limit of quantification ranged from 0.25 fmol/L to 5nmol/L.Using this method,we achieved ultrasensitive and accurate quantification of di/tripeptides in 147 plasma,49 urine and 46 bile samples obtained from healthy individuals and patients with biliary tract diseases.The iden-tified differential di/tripeptide biomarker panels showed promising diagnostic performance for patients with biliary tract cancer with area under the receiver operating curve values from 0.870 to 0.996.Further-more,this method was successfully applied to quantify di/tripeptides in the extract of an animal-derived traditional Chinese medicine,Eupolyphaga sinensis Walker.These findings highlight the possible appli-cation of the analytical method in clinics and for the purposes of quality control of traditional Chinese medicines.
维普
万方数据