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.

Characterization of natural peptides in Pheretima by integrating proteogenomics and label-free peptidomics

Pheretima,also called“earthworms”,is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edition).However,its zoological origin is unclear,both in the herbal market and CPMs.In this study,a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms(restricted search,open search,and de novo)was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima,including Pheretima aspergillum(PA),Pheretima vulgaris(PV),and Metaphire magna(MM).We identified 10,477 natural peptides in the PA,7,451 in PV,and 5,896 in MM samples.Five specific signature peptides were screened and then validated using synthetic peptides;these demonstrated robust specificity for the authentication of PA,PV,and MM.Finally,all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills,revealing the inconsistent Pheretima species used in these CPMs.In conclusion,our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines,especially non-model species with poorly annotated protein databases.

Chinese herbal medicines with beneficial effects

Among the vast resources of traditional Chinese medicine(TCM)herbal species,only a handful of Chinese herbs are growing in frigid regions or extreme environment but they have a unique property.The most recognizable TCM herb falling in this category is Panax ginseng,which is widely considered the representative tonic herb with oceans of beneficial effects on human health.In this article,we will introduce several typical Chinese herbal medicines with beneficial effects aiming to arouse broader attention from the scientific community to expand the exploration and exploitation on this for their potential applications to meet the increasingly demanding medical needs.