Detection Of Cytosine Modifications In HIV Infected Cells By Chemical Labeling Coupled With LC-MS

Objective:A highly sensitive LC-MS(liquid chromatography-mass spectrometry)method was developed for the analysis of cytosine 5-methylation on DNA/RNA and the modification of its oxidized derivatives.This method was used for qualitative and quantitative analysis of DNA/RNA cytosine 5-methylation and oxidative derivative modification in THP-1 cells and TZM-bl cells infected with HIV-1 and uninfected with HIV-1,so as to laid a foundation for further study of the influence and role of epigenetic modification in HIV-1virus infection.Methods:We selected BDEPE(2-bromo-1-(4-diethylamino-phenyl)-ethanone)as the derivatization reagent to mark DNA/RNA cytosine5-methylation and oxidative derivative modification,so as to improve the response of the target analyte in mass spectrometry detection and carry out qualitative and quantitative analysis of cytosine modification changes of cells infected with HIV-1 and uninfected with HIV-1.First of all,we optimized the derivatization conditions of 5-md C(5-methyl deoxycytidine),5-mr C(5-methylnon-deoxycytidine)and 5-mr Cm(2-oxmethyl-5-methylnon-deoxycytidine)with BDEPE as the modified representative,and obtained the most suitable reaction conditions.At the same time,the conditions of LC-MS for the detection of target products were optimized,and a highly sensitive analytical method for cytosine modified chemical labeling combined with LC-MS was established.Then,HIV-1 IIIB-TZM-bl cell infection model and HIV-1-Bal-THP-1 cell infection model were established.Cell samples were collected 3days after hiv-1 infection,and total DNA and RNA were extracted by Trizol method.Then,a certain amount of DNA and RNA were taken for enzymatic hydrolysis into nucleosides.After the enzymatic hydrolysis sample was desalted by SPE,the derivative reaction was conducted with the derivative reagent BDEPE,respectively.At last,the high sensitive qualitative and quantitative analysis of DNA and RNA cytosine methylation and oxidative derivative modification of the two cell models were carried out by using UPLC-QTOF(Ultra high performance liquid chromatography-Quadrupole tandem time of flight mass spectrometry)and UPLC-Qq Q(Ultra high performance liquid chromatography-Triple quadrupole mass spectrometry).Finally,T-test was used to analyze and compare the changes of cytosine content before and after HIV-1 infection.Results:1 Establishment of the analytical method of chemical labeling combined with LC-MS:1.1 The optimal reaction conditions of the derivative reagent BDEPE and the DNA/RNA modified nucleosides were:The reaction system of 200μL ACN(acetonitrile)contains 2m M derivatization reagent bdepe,4m M TEA(catalyst triethylamine)and reaction substrate,which react in water bath at 60℃for six hour.The conversion rate of derivatization is OK,the conversion rate of various modifications is over 90%,and the derivative products are stable within 48hours after reaction.1.2 GCB-SPE(Solid phase extraction desalination):when DNA/RNA is hydrolyzed to nucleoside,it needs to be desalted by GCB-SPE before it can react with BDEPE for derivatization.After calculation,the recovery rate of the target substance after desalting by this method is over 85%,which shows that the recovery rate of the target substance after GCB-SPE is OK.1.3 Liquid method:By comparing different mobile phase conditions,we choose 0.05%、0.1%formic acid solution(v:v)and acetonitrile solution as mobile phase A and B.After the liquid phase separation,we use the full scanning mode of high-resolution mass spectrometry to acquire the accurate molecular weight of the target product.By comparing with the retention time and the accurate molecular weight of the target product,we can determine the quality of the target product of the actual sample.Then,the MRM(multiple-reaction monitoring)model of Qq Q(triple quadrupole mass spectrometry)was used to quantify the labeled products of 12 targets in each sample.1.4 Chemical labeling combined with LC-MS:the sensitivity of modified nucleosides in the liquid was increased by 5-666 times by bdepe derivatization.Only 3 kinds of modified nucleosides,5-mr C,5-fr Cm and 5-hmd C,were detected in the cell samples without derivatization.After derivatization,12 kinds of modified nucleosides could be detected in the cell samples.And 5-md C,5-hmr C,5-fd C,5-cad C,5-mr C and 5-mr Cm have a good linear trend in the range of 1/10~6-250/10~6(/10~6G),and their correlation coefficients(R)are higher than 0.99,so we can carry out accurate quantitative analysis on these modifications.2 Detection results of cytosine modification before and after HIV-1infection:2.1 The results of modification of intracellular cytosine and its oxidized derivatives 3 days after HIV-1 Bal strain infected THP-1 cells showed:compared with the control group,after HIV-1 infection,the content of most of the target modifiers decreased in varying degrees,in which the content of 5-md C decreased from 2.842 modifiers/10~6d G to 0.106 modifiers/10~6d G,about 27times lower.The content of 5-hmrcm decreased from 0.477 modified/10~6r G to0.043 modified/10~6r G,about 11 times lower.The T-test analysis showed that there were significant differences in the down-regulation of 5-md C,5-hmd C,5-fd C,5-mr C,5-fr C,5-hmr Cm and 5-fr Cm between the two groups.2.2 The results of modification of intracellular cytosine and its oxidized derivatives 3 days after HIV-1 IIIB strain infected TZM-bl cells showed:compared with the control group,the infected group was similar to THP-1 cells after HIV-1 infection,and most of the modified contents were reduced in varying degrees.The results of T-test showed that the contents of 5-md C,5-hmd C,5-hmr C,5-fr C,5-hmr Cm and 5-fr Cm in the treated group were significantly different from those in the control group.Conclusions:In this study,we established a method for the detection of cytosine modified nucleosides by chemical derivatization labeling combined with liquid chromatography-mass spectrometry,which improved the sensitivity of mass spectrometry for the detection of cytosine modified nucleosides.Twelve target modifications of cytosine were detected simultaneously on DNA and RNA of cell samples,and this method is suitable for the detection and analysis of cytosine modified nucleosides on DNA and RNA of different biological samples.Using this method,we quantitatively analyzed the methylation and oxidative modification of cytosine 5 in two different cell lines infected with HIV-1 virus.By comparing the content of 12 cytosine modifications in the infected group with that in the control group,it was found that HIV-1 IIIB and HIV-1 Bal strains infected with TZM-bl and THP-1 cell lines,respectively,would cause a significant downregulation of most cytosine modifications in DNA and RNA,It is suggested that HIV-1 infection can cause changes in cytosine modification level,which may affect the expression and regulation of some genes,and lay a foundation for further exploring the physiological role of cytosine modification in the process of HIV-1 infection and replication.