The Development Of Pretreatment Methods For Target Low Abundance Proteins In Serum By Mass Spectrometry

With the completion of human genome sequencing,the annotation and functional confirmation of human genes have become one of the most important tasks facing life sciences.The in-depth study on the functional executors of life activities,protein,not only helps to reveal the essence of life activities in a panoramic manner,but also has important significance for studying disease mechanisms,developing early warning,diagnosis and treatment methods.The accurate analysis of low-abundance proteins in complex matrices has always been the focus and difficulty of protein research.Recently,mass spectrometry has become a powerful tool for accurate analysis of low-abundance proteins in complex matrices.However,due to the interference from other high-abundance proteins,the direct mass spectrometry analysis is difficult.Therefore,the development of enrichment materials and separations purification strategy for low-abundance proteins is an important foundation for mass spectrometry analysis.To solve the above problems,this paper took low abundance proteins heat shock protein 90α（Hsp90α）and human growth hormone（h GH）in serum as the research objects,and carried out the following research work:A novel immunoaffinity material Mo S₂@Fe₃O₄@Au NPs@DNA TET@Ab based on DNA tetrahedron was prepared for enrichment of low abundance protein Hsp90αin serum.Based on the characteristics of DNA tetrahedrons with excellent biocompatibility and regular distribution of modified groups,a new immunoaffinity material Mo S₂@Fe₃O₄@Au NPs@DNA TET@Ab was designed and synthesized.Among them,molybdenum disulfide has the characteristics of good biocompatibility,large surface area,ultra-thin sheet structure,and contains a large amount of sulfur elements,which is easy to be functionalized,so as to serve as a matrix material for immunoaffinity materials.Fe₃O₄could be separated rapidly in solution with the extra magnetic field.Magnetic separation has the characteristics of saving time and effort,less loss of antibody and antigen,and improves the efficiency of the enrichment and mass spectrometry identification.Modified DNA tetrahedron is easy to be stably immobilized on the surface of the nanomaterial,and can effectively control the orientation and the distance of the antibodies,improving the efficiency of antigen capture.The newly developed immunoaffinity materials were characterized through scanning electron microscope,transmission electron microscope,energy spectrometer and superconducting interference quantum meter and so on.The experimental results proved that the material was successfully synthesized and has strong magnetic properties,which satisfied the requirement of rapid separation from solution in the extra magnetic field.The synthetic immunoaffinity materials provide a reliable separation tool for the subsequent enrichment and mass spectrometry analysis of Hsp90α,and also prove the application potential of DNA tetrahedron-based immunoaffinity materials.A mass spectrometry method based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF）was developed for the MS analysis of Hsp90α.To investigate the enrichment effect,specificity,selectivity and sensitivity of the prepared immunoaffinity materials,MALDI-TOF was used to detect the tryptic digest of Hsp90αafter enrichment by immunoaffinity materials.The experimental results showed that the new immunoaffinity material has the characteristics of high specific enrichment.After enrichment by immunoaffinity materials,we detected 17 peptides from 3μg of Hsp90αprotein.For the mixture of Hsp90αand BSA（1:1000）,9 peaks corresponding to Hsp90αprotein were observed in the mass spectra.When the concentration of Hsp90αwas as low as 10 ng/m L,6peptides of Hsp90αwere still identified.Finally,11 tryptic peptides of Hsp90αfrom the samples of cancer patients were successfully observed.The above results show that the immunoaffinity material has high specificity,selectivity and sensitivity.The analysis strategy has broad application prospects in the accurate detection of tumor biomarkers in clinic.A mass spectrometry method based on off-line two-dimensional high performance liquid chromatography for separating serum samples was developed to accurately and quantitatively analyze the low-abundance human growth hormone content in human serum.Isotopically labeled human growth hormone was used as an internal standard.A strategy for separation of serum samples based on off-line two-dimensional high-performance liquid chromatography（2D HPLC）was established.Then,combined with high performance liquid chromatography-isotope dilution mass spectrometry（IDMS）method,quantitative analysis of low-abundance protein growth hormone in human serum was performed with high accuracy.Based on the measurement and uncertainty calculation of the simulated samples（blank serum with known quality of growth hormone standard substance,theoretical concentration 12.00 ng/g）and international comparison samples,the quantitative results are（11.45±2.33）ng/g and（12.84±1.46）ng/g,respectively.This method has high accuracy and good repeatability.In conclusion,an effective measurement method for accurate quantitative analysis of low-abundance proteins in complex matrices has been developed and the results could be traced to reference material.