Structural Characterization And Quantitative Detection Of Cyclic Peptides Based On High Performance Liquid Chromatography-orbitrap Super-resolution Mass Spectrometry

Cyclic peptides are an important class of peptide compounds with a cyclic structure and higher stability than linear peptides.It has potential in many fields,especially in the field of biomedicine.In recent decades,natural cyclic peptides with biological activity have been continuously discovered.There are more and more technical methods used to study cyclic peptides,but the content of cyclic peptides in natural species is usually less,and there are more species with similar structures and many modifications.The difficulty of separation and purification has brought obstacle to discover and identify new species.On the other hand,the lack of standards has also led to the fact that traditional liquid phase chromatography methods that rely on comparison retention time to identify species cannot be used on most cyclic peptides and cannot meet the requirements of rapid detection.This work has made a preliminary attempt to study cyclic peptides by high performance liquid chromatography combined with multistage mass spectrometry without relying on standards.The specific research contents are as follows:1.Firstly,high-resolution mass spectrometry was used to explore the applicability of the bottom-up method in the characterization of cyclic peptides.Bottom-up method requires cyclic peptides with abundant restriction sites,and the enzymatic hydrolysis is not complete.For cyclic peptides containing cysteine,reductive alkylation is required,which is complicated and time-consuming.Therefore,we mainly explored the fragmentation characteristics of different fragmentation modes in the structure characterization of cyclic peptides in the top-down method.Using collision-induced dissociation(CID)and high-energy collision-induced dissociation(HCD)two fragmentation modes to characterize the structure of small cyclic peptides,it is found that low-valence ions are CID fragmentation mode can produce fragments with more complete sequence,which is beneficial to directly obtain sequence structure.High-valence ions can generate more fragments in HCD fragmentation mode,and the two can achieve fragment information complementarity.Further characterization of the macrocyclic peptides revealed that the three valence did not reach the applicable scope of the electron-transfer dissociation(ETD)fragmentation mode.The EThcd fragmentation mode can provide a wealth of fragmentation information.2.On the basis of the above conclusions,high performance liquid chromatography combined with multistage mass spectrometry was used to analyze and detect the extract of the commercial chinese medicine Vaccaria segetalis.The chromatographic and mass spectrometry conditions were optimized,and five cyclic peptides in the extract were preliminarily determined through accurate molecular weight comparison.Based on the cyclic peptide component of Vaccaria segetalis extract forming a molecular ion peak with charge of one in the mass spectrum,CID fragmentation mode of tandem mass spectrometry(tandem MS)was selected to characterize the five cyclic peptides,and the fragment information was compared.The five cyclic peptides are identified as segetalins A,segetalins B,segetalins D,segetalins E,and segetalins I.By the retention time of the self-purified reference substance of high performance liquid chromatography,five cyclic peptides has been further verified.3.Based on the previous work,it has been extended.High performance liquid chromatography combined with multistage mass spectrometry has been further applied to the analysis and detection of cyclic peptides in commercial flaxseed extracts.The identification and quantification methods were established through optimization of analysis conditions: by accurate molecular weight determination and tandem MS analysis,16 and21 flaxseed cyclic peptides were identified in raw and cooked flaxseed extracts,respectively.The degree of oxidation was quantitatively characterized by the ratio of the integrated peak area of the oxidized cyclic peptide and the added standard cyclic peptide.The results show that the high-temperature drying step has a significant effect on the oxidation of flaxseed cyclic peptides.The presence of oxygen is conducive to the oxidation of methionine residues;the oxidation level of brown flaxseed is more sensitive to temperature and oxygen conditions.It provides qualitative and quantitative indicators at the molecular level for the quality evaluation of linseed oil,and provides a program reference for the quality evaluation of the same type of commercial food industrial products.