Mass Spectral Data Processing And Application In The Potential Biomarker Of Non-alcoholic Fatty Liver Disease

This topic is aimed at the problem of poor repeatability of low-flow mass spectrometry.This work starts from the practical problem of non-alcoholic fatty liver disease affecting factor-fibroblast growth factor(FGF-21).Combined with raw data processing algorithms,high-flow high-resolution mass spectrometry,as well as nontargeted and targeted quantitative proteomics techniques are used to achieve relative and absolute quantification of FGF-21 proteins and peptides.A new experimental strategy and data support for clinical diagnosis and biomarker discovery of non-alcoholic fatty liver disease are provided.The main works of this subject is as follows:(1)Study on chromatographic overlapping peak separation algorithm.Aiming to the chromatograph existing overlapped peaks that impacted the quantitative analysis,the method of overlapped peak resolution based on forward-backward fitting was researched.To verify the effectiveness of the forward-backward fitting algorithm,this paper designed the simulation experiments with different overlap degrees and the separation experiments of pxylene and m-xylene measured by gas chromatography mass spectrometry.This paper compared forward-backward fitting method with perpendicular-drop,intersection vertical and proportional distribution methods.Results show that maximal error of three methods reached to 29.92% and the error of the proposed method was less than 1.8%.Therefore,the forward-backward fitting method has certain quantitative analysis advantages.(2)Study on mass spectral library pre-search algorithm.In order to solve the problems of long retrieval time,large retrieval space and low retrieval accuracy of mass spectral library search algorithm,a pre-retrieval optimization algorithm based on molecular ion characteristic peaks and fragment ion characteristic peaks of mass spectrometry was proposed.The algorithm mainly includes three stages: feature peaks selection,fuzzy positioning and precise positioning.To evaluate the performance of the proposed algorithm,a series of experiments are designed by using main library and replicate library of NIST11 as reference library and query library,respectively.Moreover,real application data are used to verify the performance of FPP method.Compared to two-step spectral library pre-search(TSLP)algorithm and "ten-peak" method,the results show that FPP method can obtain higher accuracy,smaller pre-search space,shorter time consumption and less remaining spectra.(3)Relative quantification of serum target proteins based on large flow mass spectrometry.Because specific proteins associated with disease in blood are usually diluted to the concentration range of ng/m L,it is difficult to detect them.Low-flow mass spectrometry can be used to detect them,but the repeatability is not good.Based on this situation,in order to explore the content of affecting factor(fibroblast growth factor,FGF-21)of non-alcoholic fatty liver disease in serum,an experimental strategy based on high-flow mass spectrometry was proposed to detect the limit of detection.The results showed that the signal intensity of precursor ion was linearly correlated with the different concentration of FGF-21.Therefore,the minimum detection limit of FGF-21 in serum was 400 pg/m L.(4)Absolute quantification of serum target protein based on parallel reaction monitoring(PRM)technology.In order to further determine the detection limit of FGF-21 protein in serum,another non-shared heavy isotopelabeled peptide of FGF-21 was synthesized.The equivalent heavy isotope-labeled peptide solution was added to the mixed enzymatic hydrolysis solution of serum and fibroblast growth factor FGF-21 protein with different concentrations.Parallel reaction monitoring(PRM),a targeted quantitative proteomics technique with high resolution and accuracy,was used for detection and analysis.The heavy and light ion chromatographic peaks of target peptide segments were extracted by Skyline software.At the same time,extract ion chromatogram of two peptides are integrated.The corresponding content of light-labeled peptide of FGF-21 were obtained by comparing the areas of extract ion chromatogram.Finally,the limit of detection limit was determined to be 400 pg/m L.