Identification Of Collagen And Glycosaminoglycans From Traditional Chinese Medicine Colla Corii Asini And Study Of The Interaction Between Glycosaminoglycans And Chemokines By Mass Spectrometry

Glue traditional Chinese medicine is an important component of animal traditional Chinese medicine,and the production scale and market sales of Colla Corii Asini(CCA)are leading.The pharmacological effect of CCA is mainly to improve metabolic balance,nourish blood,and regulate immune function.The biomacromolecules in CCA are mainly proteins and Glycosaminoglycan(GAG).At present,the research on donkey-hide gelatin is not comprehensive enough,so the first goal of this paper is to study the structure of biomacromolecules in donkey-hide gelatin,study the composition of proteins and GAG in skin and gel samples,and establish relevant mass spectrometry methods.The interaction between GAG and protein plays an important role in the organism.In the research of CCA,we analyzed the protein sequence and GAG sugar unit,but the interaction between GAG and protein requires more complex mass spectrometry and other new technologies.The mass spectrometry method established by CCA can perform preliminary analysis on proteins and GAG,including protein sequence analysis and sugar unit structure identification,while the qualitative and quantitative characterization of sequence length,types of glycosylation,sugar chains and glycosylation sites of complex glycoprotein mixtures requires further method development.Therefore,the remainder of this article focuses on the development of more complex analysis methods of glycoprotein isoforms and the study of the interaction between GAG and chemokines combined with Bio-layer Interferometry(BLI)and affinity column technology.The interaction between GAG and chemokines is related to many physiological and pathological processes,so we chose CCL5 as a model molecule to establish a correlation analysis method.CCL5 is a member of the chemokine family.Chemokines refer to some low-molecular-weight(6-14 kD a)basic proteins,which play an important role in the inflammatory response due to their targeted chemotaxis attracting leukocytes to the site of infection.CCL5 is also called regulated on activation normal T cell expression and secretion(RANTES),and belongs to CC chemokine subfamily(?-type chemokine).The binding of CCL5 to its receptor can inhibit the entry of HIV virus into cells,but due to the effect of inflammatory effects,modifying the modification of CCL5 can change the inflammatory effect without affecting its effect of inhibiting virus entry.The combination of CCL5 and GAG is a prerequisite for CCL5 to form a chemokine gradient and exert its chemotaxis properties.It is also a prerequisite for the interaction between CCL5 and CCR receptors.Therefore,the research on the interaction between CCL5 and GAG is of great significance.To solve the above problems,first,we must understand the structure and composition of natural CCL5 in the human body.Secondly,according to the literature,15 different amino acid truncated and glycosylated CCL5 isoforms were synthesized,and corresponding qualitative and quantitative analysis methods were established to study the interaction of different CCL5 and GAG in different situations.The established method was used to determine the interaction between different CCL5 and GAG,and to explore and explain the interaction mechanism.The main research results of this paper are as follows:1.Established a method for identifying the source of CCA.The main protein of CCA was analyzed and a method for identifying the source of CCA was established.Through shotgun proteomics,the skin-like proteins of donkeys,horses,cows and pigs of different species were studied,and the main protein was identified as collagen.By adding a hydroxylation modification to search the database of the corresponding species,it was found that collagen was severely hydroxylated.The main component collagen was selected as the research object,and the type I collagen sequences of different species were compared by bioinformatics to infer the theoretical characteristic sequence of each species.Mass spectrometry was performed on the skin-like enzymatic sample,the parent ion of the theoretical characteristic sequence was extracted,the characteristic peptide of each species was determined according to the screening conditions,and the corresponding characteristic peptide was extracted from the gel-like sample,thereby confirming the feasibility of the characteristic peptide.The corresponding characteristic peptide was synthesized and the Multiple Reaction Monitoring(MRM)method for detecting the corresponding characteristic peptide was established to improve the method sensitivity.The method was validated and applied by analyzing adulterated samples and commercial gelatin.Eventually,0.5%adulterated CCA can be detected by this method,and this method can be used to identify mules of the hybrid species of donkey and horse,thereby excluding the possibility of adulteration.2.Analyzed the GAG changes during donkey skin degradation.After degreasing the donkey skin and gelatin hydrolysate,proteolysis was performed to remove most of the proteins,and then GAG were collected through a cation exchange column,The extracted GAG were completely digested into disaccharides using heparinase ?,?,? and CSABC enzymes.By establishing the LC-MRM-MS/MS method of disaccharides,the peak areas of the di-extracted ion peaks of standards and samples were integrated to obtain the composition and content of disaccharides in donkey skin and CCA.We have a basic understanding of the role of GAG in the process of making donkey skin as gelatin.It is found that CS is more abundant and stable in gelatin than HS.It is speculated that it may play a more important role in the efficacy of gelatin effect.3.Identified natural CCL5 in human plasma.The literature reports that CCL5 has two possible O-glycosylation sites and three different amino acid lengths from 1-68,3-68,and 4-68.Therefore,we established a method to identify the natural form of CCL5 in human plasma.The content of CCL5 in plasma was extremely low,so firstly a homemade multi-antibody agarose gel affinity column of CCL5 was used to enrich CCL,in human plasma and analyzed by mass spectrometry.In the end we identified three forms of CCL5 with different amino acid lengths.For the presence of O-glycosylation in CCL5 in plasma,O-GalNAc glycopeptides in the sample were enriched using a homemade VVA lectin affinity column,and an internal standard isotope glycopeptide of 5%of the theoretical protein was added.The identification,of the added internal standard glycopeptide verified the feasibility of the method,but no related glycopeptide of glycosylated CCL5 was identified,indicating that the glycosylated form of CCL5 in human plasma was less than 5%.In the future,this method can be used to analyze biological samples with high CCL5 content,so as to characterize the glycosylation structure of CCL5.4.Established a qualitative and quantitative method for identifying CCL5 isoforms.Due to the extremely low content of CCL5 in human plasma,15 types of glycosylated CCL5 were designed and chemically synthesized based on the reported forms of CCL5 in the literature,including different amino acid lengths,different glycosylation,and different glycosylation sites.The difficulty in identifying the 15 CCL5 isoforms is that there are 6 groups of isomers with only different glycosylation sites.Due to the small differences of these 6 isomers,the column cannot be distinguished,and Electron Transfer Dissociation(ETD)fragmentation can preserve the integrity of the sugar chain and generate characteristic c/z fragment ions?The external standard method was selected as the quantitative method.The m/z of the sequence determined by Higher Energy Collision Induced Dissociation(HCD)fragmentation was divided into 9 groups,and then 6 groups were quantified by characteristic fragment ions during ETD fragmentation.Therefore,we combined the HCD and ETD fragmentation methods of mass spectrometry to identify and quantify all CCL5 isoforms,so as to establish a method for distinguishing isomeric glycoproteins with minor differences.This method was first used to analyze the eluent of a mixture of CCL5-1 and CCL5-7 after passing through a heparin column.CCL5-1 is a 1-68 sequence length and non-glycosylated CCL5,CCL5-7 is a 3-68 sequence length and Ser4 has GalNAc,so the experimental results show that the interaction between CCL5 with different structures and heparin is different,which is follow-up research established the basis.5.Explored the interaction mechanism of CCL5 and heparin.In order to investigate the interaction between a single CCL5 and heparin,the biofilm interference technology was used to analyze the interaction between each CCL5 and heparin using the principle of light interference to determine different KD values.According to the measurement principle of BLI,the smaller the KD value is,the closer the interaction is,and the larger the affinity effect is.The combination analysis of CCL5 structure and KD value showed that for the three non-glycosylated isoforms,when two amino acid residues were truncated,the binding affinity decreased slightly,and if one amino acid residue was missing Then the binding affinity increases slightly,indicating that the truncation has little effect on the affinity.The Ser-5 position is more critical than the Ser-4 position.In terms of glycan structure,disaccharide residues generally have a stronger and noticeable effect on heparin binding than monosaccharide residues.The software was used to simulate the molecular docking of CCL5-11,CCL5-14 and CCL5-15 with heparin octose.It was found that Ser5 and disaccharide glycosylation affected the binding sites of CCL5 and heparin and the number of basic amino acids,which affected the strength of their interaction.In order to investigate the interaction between CCL5 and heparin under mixed conditions,different CCL5 was mixed through a heparin affinity column,and the eluent at the peak was collected.The established method was used for qualitative and quantitative analysis.The difference in the elution amount of each CCL5 combined with its structural analysis,the results show that different amino acid sequence changes,different positions and types of glycosylation modifications can have various effects on heparin affinity.At the same time,in different combinations,the relative affinities of the various isoforms remained the same.It shows that the effect of a certain modification on the affinity of CCL5 isoforms and heparin is stable,not affected by other isoforms or different mixture compositions,but the results are not the same as those of the individual action.According to the above results,it is shown that CCL5 Ser5 glycosylation site with disaccharide glycosylation modification has a greater impact on the nature of the interaction between CCL5 and heparin,whether it is combined alone or mixed.These results provide us with guidance on how to alter the biological activity of CCL5 by specifically designing glycosylated isoforms.Based on this paper,we first have established a mass spectrometry method to study collagen and GAG of traditional Chinese medicine donkey-hide gelatin,and developed a characteristic peptide method for the source identification of donkey-hide gelatin(the supplementary inspection method of donkey-hide gelatin has been applied by Shandong Food and Drug Inspection Institute for Chinese Pharmacopoeia),which can fake 0.5%of the sample and distinguish between donkeys,horses and mules.Then the composition and content of GAG sugar unit in CCA were analyzed.The interaction between GAG and protein is related to a variety of physiological processes in vivo.We selected the interaction between heparin and CCL5 to study.Natural CCL5 in human plasma was analyzed using mass spectrometry established by CCA.The content of natural CCL5 is too low,in order to analyze the complex glycoprotein mixture and study the interaction between GAG and CCL5,we synthesized 15 CCL5 isoforms with differences in sequence length,sugar chain and glycosylation site,and identified them in CCA The analysis method of CCL5 isoform mixture was further developed on the basis of the sequence and sugar unit analysis methods accumulated in.The affinity constant of heparin and each CCL5 was determined by BLI,and the developed mass spectrometric analysis method was used to qualitatively quantify the eluent of different mixed forms of CCL5 passing through the heparin column.Based on the results of BLI analysis,we found that the structure of Ser5 with disaccharide has a greater influence on the interaction between CCL5 and heparin.