Development of Chemical Isotope Labeling Liquid Chromatography Orbitrap Mass Spectrometry for Comprehensive Analysis of Dipeptides

Dipeptides have recently attracted considerable attention
due to
their newly found biological functions and potential biomarkers of
diseases. Global analysis of dipeptides (400 common dipeptides in
total number) in samples of complex matrices would enable functional
studies of dipeptides and biomarker discovery. In this work, we report
a method for high-coverage detection and accurate relative quantification
of dipeptides. This method is based on differential chemical isotope
labeling (CIL) of dipeptides with dansylation and liquid chromatography
Orbitrap tandem mass spectrometry (LC-Orbitrap-MS). An optimized LC
gradient ensured the separation of dansyl-dipeptides, including positional
isomers (e.g., leucine- and isoleucine-containing dipeptides). MS/MS
collision energy in Orbitrap MS was optimized to provide characteristic
fragment ion information to sequence dansyl-dipeptides. Using the
optimized conditions, a CIL standard library consisting of retention
time, MS, and MS/MS information of a whole set of 400 dansyl-dipeptides
was constructed to facilitate rapid dipeptide identification. For
qualitative analysis of dipeptides in real samples, IsoMS data processing
software’s parameters were tuned to improve the coverage of
dipeptide annotation. Data-dependent acquisition was also carried
out to improve the reliability of dipeptide identification. As examples
of applications, we successfully identified a total of 321 dipeptides
in rice wines and 105 dipeptides in human serum samples. For quantitative
analysis, we demonstrated that the intensity ratios of the peak pairs
from 96% of the dansyl-dipeptides detectable in a 1:1 mixture of 12C- and 13C-labeled rice wine samples were within
±20% of an expected value of 1.0. More than 90% of dipeptides
were detected with a relative standard deviation of less than 10%,
showing good performance of relative quantification.