| BackgroundCytochrome P450 enzymes(CYP450)and uridine diphosphate glucuronic acid transferases(UGTs)that mainly exist in the liver are involved in the metabolism of endogenous and exogenous chemicals and drugs.CYP450 and UGTs participate in I phase reaction and II phase reaction respectively.Cytochrome P450 oxidoreductase(POR)and cytochrome b5(b5)which provided electrons to drug metabolizing enzymes also play an important role in drug metabolism.Polymorphisms and content differences of the CYP450 and UGTs are the major factors that result in differences between individuals in drug therapy.Hepatocellular carcinoma(HCC)is one of the most common tumors.Clinical dosage needs to adjust in time in HCC patient.However,as the basis of dose adjustment,how the content of CYP and UGT in HCC patients changes is still not entirely clear.Therefore,it is extremely important in clinical individualized treatment to quantify the content of drug-metabolizing enzymes in cirrhosis.Western Blotting,ELISA and mass spectrometry were employed to detect the content of the drug-metabolizing enzymes.Based on binding reaction of antigen and antibody,Western Blotting and ELISA are sensitive and specific but their applications are restricted,because it is difficult to prepare specific antibodies and to simultaneously quantify various proteins.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)has won many concerns day by day and has been applied in the determination of drug metabolizing enzymes with a wide dynamic range,high sensibility and throughput.In this paper,liquid chromatography parallel reaction monitoring mass spectrometry(LC-PRM/MS,PRM)was used to quantify the content of the drug-metabolizing enzymes of cirrhotic tissue in liver that can be used as a reference for hepatocirrhosis patients in the clinical medication in the future.Compared with the selective reaction monitoring mass spectrometry(SRM)method,LC-PRM/MS can obtain all fragments,and the results could ensure quantitative specificity.This thesis consists of two parts.In the first part,the stable isotope labeling technique was used to prepare a multiplexed quantification concatemer(Qcon CAT).And in the second part,the PRM method was established and applied to the quantification of drug metabolizing enzymes.Method1.Preparation of the internal standard peptide 2-3 specific peptides of each of the twenty-two kinds of drug-metabolizing enzymes were seclected.The tryptic peptides making up the Qcon CAT were expressed by Escherichia coli in SILAC Medium.The recombinant protein was purified through Glutathione Sepharose 4B and the purity was determined by SDS-PAGE gel scan analysis.Then trypsin enzyme was used to digest the recombinant protein.2.Investigation of the efficiency of cutting and tagging Data-dependent acquisition mode was used to investigate the cutting and tagging efficiency and the results were characterized by Proteome Discoverer 2.1.3.Quantification of the internal standard peptide Standard peptide ASGNLIPQEK from CYP1A2 was synthesized and quantified by the amino acid hydrolysis method.Standard peptide was diluted into serial concentration gradient and added the same amount of internal standard peptide.The content of internal standard peptide was detected by LC-MS/MS.4.Quantification and digestion of liver microsomes Human liver microsomes(HLMs)were prepared by differential velocity centrifugation.Total protein concentrations were measured by Bradford method.10 μg of the protein was removed after quantification and was digested by trypsin for 26 hours.5.The establishment of a liquid chromatography-mass spectrometry parallel reaction monitoring method Internal standard peptide was mixed with digested human liver microsomes.Data-dependent acquisition mode was used to confirm with three pre-columns.Proteome Discoverer 2.1 library was used to identify the results to determine the retention time and the limit of quantitation and product ion for the target peptide.6.Establishment of standard curves The Qcon CAT protein was diluted into serial concentration gradient and added the same amount of trypic human liver microsomes.Liquid chromatography-parallel reaction monitoring mass spectrometry was used to establish the standard curves.7.Application of liquid chromatography-mass spectrometry parallel reaction monitoring method The PRM was applied to the determination of drug metabolizing enzyme content in cirrhosis tissue of liver cancer patients.Results1.Identification of the purification and quantification of the recombinant protein SDS-PAGE analysis showed that the recombinant protein was highly purified.The recombinant protein was quantified using the Bradford method,and the results showed that the linear equation was y=9.8617x+0.1537,R2 was 0.9876 and the protein concentration was 0.56 mg/m L.2.Qualitative analysis of recombinant protein Proteome Discoverer 2.1 was used to match the target protein and the results showed that internal standard peptides well match with the target protein.3.Cutting and labeling efficiency of recombinant protein The Qcon CAT had high efficiency of digestion and the results showed that the percentage of missed cleavage was under 10%.Each peptide was labeled equably and the labeling efficiency was about 90%.4.Absolute Quantification of Recombinant Proteins The average of the fragment y5 and y4 was 134.75 fmol and was used as the quantitative result of Qcon CAT.Compared with the results of precursor,the difference was less 1.1-fold.5.Determination of retention time and quantitative fragments The retention time of the same peptide had deviation when separated with the different pre-columns,but the deviation was always less than one minute when the same pre-column was used.The three fragments with the highest intensity were used to quantify the peptide.6.Establishment of standard curves Liquid chromatography-parallel reaction monitoring mass spectrometry was used to established standard curves and the results showed that R2 values were above 0.98 and the dynamic ranged from 5fmol to 200 fmol.7.Quantification of drug-metabolizing enzymes in cirrhosis tissue in patients with hepatocellular carcinoma The contents of six drug-metabolizing enzymes in 12 samples were determined.The results showed that the content of CYP1A2,UGT1A1,UGT2B4,UGT2B15,POR and b5 were 67.36 fmol/μg、87.25 fmol/μg 、 92.97 fmol/μg 、 53.01 fmol/μg 、 140.65 fmol/μg and 84.91 fmol/μg,respectively.And the interindividual variability were 6.5,5.02,5.24,4.33,6.17 and 4.02 respectively.Compared with normal tissues,POR was highly expressed in liver cirrhosis tissues of hepatocellular carcinoma,and the contents of UGT1A1 and UGT2B15 were significantly lower than those of normal tissues(p <0.05).Conclusion1.Quantification concatemer was constructed and the preparation of large-scale internal standard peptide was achieved in this study.In addition,the purity and labeling efficiency of the internal standard peptide could be ensured;2.Based on the establishment of the PRM method,standard curves of absolute quantitative were established.Most of linear relationship of the curves were 0.99,and the dynamic range were 5-200fmol;3.The amount of six metabolizing enzymes in 12 samples was determined by the established method,and the results were compared with the results of normal tissues.POR was highly expressed in liver cirrhosis tissues of hepatocellular carcinoma,and the contents of UGT1A1 and UGT2B15 were significantly lower than those of normal tissues(p <0.05). |
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