Systematic screening and structural characterization of dipeptides using offline 2D LC-LTQ-Orbitrap MS: A case study of Cordyceps sinensis

Cordyceps sinensis (C. sinensis) is a widely used and highly valuable traditional Chinese medicine. Several dipeptides have been detected in C. sinensis, but current scientific knowledge of its chemical makeup remains limited. In this study, an improved approach that integrates offline two-dimensional liquid chromatography (2D LC) separation, precursor ion list, library screening, and diagnostic ion filtering was established to systematically screen and characterize dipeptides in C. sinensis. Offline 2D LC integrating hydrophilic interaction LC and reverse phase separations was established to eliminate interference and identify the target dipeptides. A library containing the potential 400 dipeptides was created, and a precursor ion list with all theoretical precursor ions was adopted to trigger the MS/MS scan with high sensitivity. To identify dipeptides, the type and connection sequence of amino acids were determined according to the product ions. Ile and Leu residues were differentiated for the first time according to the characteristic ion at m/z 69.07. Ultimately, 170 dipeptides were identified or tentatively characterized from C. sinensis, and most are reported for the first time in this species herein. In addition, the identified dipeptides were also applied for discrimination among the three Cordyceps species, and 11 markers were identified. The obtained results provide a deeper understanding of the chemical basis of C. sinensis.