Analysis And Identification Of Proteins In Three Gelatinous Chinese Medicines

Gelatinous Chinese medicines(GCMs),made from mammalian skin or horn or reptile shell after long-term decoction during their manufacturing,have curative effect on blood deficiency,bleeding and etc.Due to the large demand for GCMs,many fake or adulterate products are sold in the market,such as swine skin gelatin,skins gelatin mixture,so it has become a hot research area in recent years.Although the quality control scheme of GCMs in prevailing Chinese Pharmacopoeia(Vol.I.2015 Ed)is becoming more and more effective,the research foundation of its specific identification is yet to be enhanced.Therefore,to conduct the related basic research and then to achieve accurate identification of them is very urgent.In this thesis,proteins in the GCMs and their original medicinal parts were analyzed in order to provide the basis for the authentication of GCMs and the investigation on their fundamental substance of bioactivities.The main parts of the study are as follows:1.Determination and comparison of protein content in GCMsIndividual GCM was dissolved into ultrapure water(1:100 solid-liquid ratio)under 85 oC waterbatch for 20 mins to prepare sample solution,and protein content in GCMs was determined by both Bradford and BCA method.The results showed that the content in GCMs(4.64%~12.74%)by Bradford assay was much lower than that by BCA assay,suggesting that the proteins in GCMs don't contain a decent number of arginine and/or aromatic residues(such as Phe,Tyr,Trp).In addition,the content of protein in different batches of Asini Corii Colla(ACC),Testudinis Carapacis ET Plastri Colla(TCPC),and Cervi Cornus Colla(CCC)had certain difference.Among them,the difference in protein content between ACC and its references was relatively minor whereas the protein content of TCPC and CCC were higher than that of their respective references.2.Determination and amino acids and collagen in GCMs17 amino acids of GCMs were determined by automatic amino acid analyzer.The results showed that kinds and content of amino acids in GCMs was abundant,especially a higher content of both Gly and Pro.PCA results showed that Gly and Pro were the common characteristic amino acids among three GCMs,indicating that their protein was mainly composed of collagen.In addition,HCA showed that commercial GCMs products could be classified into three categories.Then,L-Hyp in GCMs was determined by the pre-column derivatization-high performance liquid chromatography(HPLC),and the collagen content was calculated by the conversion coefficient.The results showed that L-Hyp content in ACC,TCPC and CCC were 9.11%,6.93% and8.06%,and their collagen content were 64.65%,76.92% and 57.23%,respectively.The content of L-Hyp and collagen in 18 batches of commercial GCMs products were of certain difference.Among them,the content of L-Hyp and collagen in ACC was similar to that in its references whereas that in TCPC and CCC were higher than that in their respective references.3.Electrophoretic analysis of protein in GCMs and its original animal partsProtein composition in gelatin,GCMs and their original animal parts was analyzed by SDS-PAGE.Those SDS-PAGE profiles showed that visual protein bands were plentiful and their molecular weights were ranged from 10 kDa to above 250 kDa,demonstrating that donkey skin,turtle shell and deer antler all had abundant and various proteins.However,the major proteins in GCMs were presented in a dispersion manner,without visible protein bands.There were obvious differences in SDS-PAGE and 2-DE profiles among GCMs,and the molecular weight of ACC protein was ranged from 15 kDa to 250 kDa or above,whereas those of TCPC and CCC were both below 50 kDa.Therefore,the difference in dispersion area may provide a reference for GCMs' identification.4.Dynamic changes of protein in original animal parts during manufacturingDynamic changes of protein in original animal parts during manufacturing were studied by electrophoretic analysis.The results showed that the protein in original animal parts(donkey skin,turtle shell and deer antler)could be quickly dissolved,supposing that they contain water-soluble protein and salt-soluble protein.Since then,with the extension of decoction time and the addition of ingredients(rock sugar,rice wine and soybean oil),the macromolecule protein in original animal parts may have complicated changes in structures(such as protein degradation,polymerization),and consequently the lanes presented in a dispersion manner.5.Trypsin digestion-MALDI-TOF/TOF-MS analysis of peptide fragments of GCMsProteins in medicinal parts were separated by SDS-PAGE,and peptides were identified after in-gel tryptic digestion,followed by MALDI-TOF/TOF-MS analysis and database matching.As a result,7 unique proteins were identified from donkey skin and 1 unique protein was identified from turtle shell.Besides,those proteins were profiled by 2-DE and subject to in-gel digestion,and peptides were identified by MALDI-TOF/TOF-MS.Consequently,another 12 unique proteins were identified from donkey skin and 6 unique proteins were identified from turtle shell.In addition,protein(50~75 kDa and nearly 25 kDa)in GCMs was digested with trypsin and peptides mixture obtained were analyzed by MALDI-TOF/TOF-MS.And then,their PMF were obtained and 32 different peptide fragments were selected,which could provide a reference for GCMs' identification.6.Collagenase digestion-NanoLC-Orbitrap MSD analysis of peptide fragments of GCMsProtein in ACC,TCPC and CCC was digested with collagenase and peptides mixture obtained were analyzed by NanoLC-Orbitrap MSD.Amino acid sequence of the peptides was all explored by Mascot or de novo sequencing using PEAKS software.And consequently,14 potential biomarkers were found,including 5potential biomarkers of ACC,5 potential biomarkers of TCPC and 4 potential biomarkers of CCC.In addition,those potential characteristic peptides were used as an input sequence for BLAST search program against nrdb.They were mostly from mammalian animals.