Comprehensive Dipeptide Profiling and Quantitation by Capillary Electrophoresis and Liquid Chromatography Coupled with Tandem Mass Spectrometry

Abstract

Dipeptides have attracted much attention as post-amino acids with physical properties and functions different from those of amino acids. However, a given dipeptide cannot be distinguished by mass spectrometry from its structural isomer with an opposite amino acid binding order unless these isomers are separated before introduction, which complicates the comprehensive analysis of dipeptides. Herein, a novel analytical platform for dipeptide analysis by capillary electrophoresis tandem mass spectrometry and liquid chromatography tandem mass spectrometry is developed. This method is used to quantitate 335 dipeptides and achieves excellent separation of structural isomers with opposite binding orders, high correlation coefficients, and low instrumental detection limits (0.088–83.1 nM). Moreover, acceptable recoveries (70–135%) are observed for most tested dipeptides in chicken liver samples spiked both before and after preparation. The developed method is also applied to the quantitation of dipeptides in the livers of mice fed different diets to detect 236 dipeptides, and the shift from a normal diet to a high-fat diet is shown to increase/decrease (p < 0.05, fold-change < 0.5) the contents of 0/29 dipeptides, respectively. The developed method is expected to facilitate the search for new dipeptide applications such as novel functional components of foods and biomarkers of diseases.

Comprehensive Dipeptide Analysis Revealed Cancer-Specific Profile in the Liver of Patients with Hepatocellular Carcinoma and Hepatitis

“…In this study, we performed a comprehensive dipeptide analysis of paired tumors and surrounding nontumor liver tissues obtained from 13 patients with HCC and three patients with metastatic liver cancer. We successfully quantified 236 dipeptides (14 overlapped) with our previously developed method using LC-MS/MS and CE-MS/MS [16]. This study reports the largest number of dipeptides detected in the liver tissue to date.…”

“…Comprehensive dipeptide analysis was performed by CE-MS/MS and LC-MS/MS, as described previously [16]. The CE-MS/MS system was composed of an Agilent 1600 CE system (Agilent Technologies, Santa Clara, CA, USA), Agilent 6490 triple quadrupole mass spectrometer, Agilent 1200 series isocratic HPLC pump, and Agilent G1607A CE-ESI-MS sprayer kit.…”

“…As post-amino acids, dipeptides are highly diverse with different physical and functional properties from amino acids [16]. In recent years, dipeptides have attracted attention as functional biomaterials, particularly as potential disease biomarkers [17].…”

“…Therefore, these isomers must be separated by chromatography before being introduced into a mass spectrometer, but it is difficult to separate many dipeptides by a single analytical method. To overcome this limitation, we recently developed a comprehensive dipeptide analytical method using liquid chromatography tandem mass spectrometry (LC-MS/MS) and capillary electrophoresis tandem mass spectrometry (CE-MS/MS), which enabled simultaneous quantitation of 335 types of dipeptides [16].…”

Effects of Dietary Food Components on Cognitive Functions in Older Adults

“…Moreover, glutathione is also known as an antioxidant for the cellular detoxification of ROS in the brain [ 45 , 204 ]. Recently, Ozawa et al developed a method for the comprehensive analysis of dipeptides and showed that the profiles of dipeptides in the body were greatly altered in diseases such as cancer [ 205 , 206 ]. Clarifying the dynamics of dipeptides in food and the body will be important to elucidate the mechanism of cognitive decline.…”