Proteomics Analysis And Ultra-sensitive Detection Of Archaeological Hairs

Animal hair is one of the most important symbols of world clothing culture.In ancient times,animal hair represented religious worship,political status,and economic strength.With the development of tomb archaeology,plenty of archaeological hairs have been unearthed from all over the world,the archaeologists have been attracted to explore the origin and trade exchanges of hairy products.Among them,the species identification and the detection of micro-traces are the two major puzzles in the hair archaeology.However,the archaeological hairs are the proteinous cultural relics,and they have experienced aging and erosion during thousands of years of burial process.Keratin content in archaeological hairs is low and the archaeological hairs are mixed with various impurities.Thus,it is urgent to develop new identification methods with high sensitivity and specificity to analyze archaeological hairs.In this thesis,proteomics technology was performed to analyze animal hair keratins with different species,and the biomarkers of different animal hair were screen out.The animals were immunized with extracting keratin as the antigen to prepare specific antibody,and the antibody was applied to the immunofluorescence microscopy(IFM),western blot,enzyme-linked immunosorbent assay,and electrochemical immunosensor,to establish proteomic and immunological analysis methods for hair samples.The established methods was then used in hair archeology to analyze the species of archaeological hairs,and to achieve ultra-sensitive detection of archaeological hairs.The main research contents are as follows:1)Scanning electron microscopy,Fourier transform infrared spectroscopy,X-ray diffraction,sodium dodecyl sulphate polyacrylamide gel electrophoresis were used to analyze the morphological characteristics,molecular structure and molecular weight of animal hairs(grey fox,raccoon dog,American mink,cape hare,and lamb).Five animal keratins were extracted by alkaline method.After enzymolysis,they were detected by high performance liquid chromatography-tandem mass spectrometry.The marker proteins A0A3Q7RKS7,Q6EIZ1(F1PTX4),U6CZM3(U6DQL8),G1SUH8,E3VW87 with super specificity and high abundance were screened through cross-analysis to be used as biomarkers of grey fox hair,raccoon dog hair,American mink hair,cape hare hair,and lamb hair,respectively.The traditional identification method and proteomics method were established for hair samples and applied to the species analysis of archaeological hairs.2)The wool keratin was extracted by reduction method as immunogen to obtain specific antibody though animal immunization.Then,the antibody was applied in immunofluorescence microscopy and western blot to detect wool samples to realize in-situ observation on fibers and keratins with different molecular weight.The binding of fibers and antibody could be directly observed when applying immunofluorescence microscopy to archaeological hairs,which established a non-destructive identification method for archaeological hairs.The western blot method was applied to archaeological hairs to directly observe the binding of different molecular weight keratins and antibodies,and to detect the target keratin of the archaeological hairs.The in-situ observation methods provide new ideas for the visual identification and analysis of archaeological hairs.3)The anti-wool keratin specific antibody was used to fabricate a label-free electrochemical immunosensor for detecting wool keratin.The limit of detection(LOD)of immunosensor is 0.90ng/m L,the quantitative detection range is 0.01-100 ?g/m L,both of which are superior than that of enzyme-linked immunoassay.Both methods can effectively identify keratin in archaeological hairs,but the electrochemical method which is more stable,can detect hair relics with lower protein content.In summary,the immunological and proteomic identification methods of archaeological hairs were established,which provided new tools for the species identification and ultra-sensitive detection of archaeological hairs,and provided pioneering ideas for the research of ancient hair trade and exchanges as well.