Study On The Comprehensive Characterization Method Of Urine And Blood Nucleosides Based On LC-MS Technolog

Endogenous nucleosides,including normal nucleosides and modified nucleosides,are the end products of RNA metabolism and potential biomarkers of cancer before the appearance of morphological abnormalities.Normal nucleosides can be phosphorylated and re-utilized to synthesize novel RNA or further degraded to form Uric acid,β-alanine andβ-aminoisobutyric acid in vivo.However,modified nucleosides cannot be reused and further metabolized due to the lack of related phosphorylase and oxidase.In addition,high levels of modifi ed nucleosides in the body have toxic effects.Therefore,modified nucleosides circulate in blood stream and are then excreted into urine.The signifi cantly increased output of urinary nucleosides has a certain relation with the increased RNA turnover and modification enzyme activity in the process of genetic information transfer.It was reported that the altered RNA turnover and increased modification enzyme activity are both triggered by cancer.Therefore,endogenous nucleosides in serum and urine are important biomarkers of various types of cancer.Currently,the comprehensive characterization of endogenous nucleosides faces a great challenge due to the low levels and high polarity of nucleosides and the complex matrix in urine and serum,especially for the discovery and identification of new nucleosides.In this study,the systematic identification of urinary and serum nucleosides was completed and comprehensive characterization of nucleosides was developed by combining UPLC Q-TOF/MS and UHPLC Q-Trap/MS.Molecular prediction provides a powerful tool for new nucleosides and other new compounds.Brief introduction to the study achievements of this study was showed as follows:1.Literature reviewThe previous reports on the source and research progress,sample preparation,and detection method of urinary and serum nucleosides have been briefly summarized in this paper.This review provides theoretical basis and methodological guidance for this study.2.Construction of modified base,modified ribose and nucleosides in-house databaseAbout 100 nucleosides have been reported in the literature by summing up researches about urinary and serum nucleosides.Nucleosides consist of base and ribose which were connected by C-N bond except pseudouridine.The most of modified nucleosides in serum and urine are modified at one of the parts(base or ribose).modified base and ribose database including predicted modified ribose were constructed in order to facilitate data processing.The in-house database includes known nucleosides,modified base,modified ribose and predicted nucleosides.At last,the molecular formula,protonated molecule and protonated base accurate mass to charge ratio of predicted nucleosides were calculated.3.Systematic identification of urinary nucleosides by UPLC Q-TOF/MSThere are 5 steps in the systematic identification of urinary nucleosides by UPLC Q-TOF/MS:study on fragmentation pathways of nucleosides,optimization of mass spectrometry parameters,optimization of liquid chromatography conditions for the separation,optimization of sample preparation conditions and comprehensive identification of urinary nucleosides.There is no systematic study for nucleosides fragmentation pathways by mass spectrometer with an electrospray ion source at the present.The fragmentation pathways of normal nucleosides and modified nucleosides have been researched in this paper through nucleoside standards and reported data in the literature.Analysis conditions,including mass spectrometry parameters,liquid chromatography conditions for the separation and sample preparation conditions,were studied in order to detect nucleosides as many as possible.First of all,cone voltage and collision energy were optimized by comparing mass spectrometry signal strengths under different conditions by UPLC Q-TOF/MS.Secondly,reverse phase and normal phase ultra-performance liquid chromatography separation were optimized.The experiment result shows that reversed-phase liquid chromatography resulted in a better separation of urinary nucleosides.Thirdly,there were 25,10,22 and 51 nucleosides which were detected respectively by UPLC Q-TOF/MS from urine prepared with protein precipitation,Oasis HLB solid phase extraction,Oasis HLB solid phase extraction and phenylboronic acid(PBA)solid phase extraction.In this study,solid phase extraction based on PBA-functionalized was chosen to selectively purify and enrich the cis-diol-containing nucleosides from urine and serum for further research.Full scan high resolution mass spectrometry(HRMS)data were acquired by UPLC Q-TOF/MS in positive ion mode.Compounds containing protonated molecules(MH+)and its protonated base(BH+)were supposed to be nucleosides or its isomers initially.To screened out nucleosides from full scan HRMS data,extracting MH+ and BH+simultaneously was conducted.Three steps for the identification of urinary nucleosides are as follows:(1)The nucleosides with standard were identified by comparing the retention time,precise molecular mass of MH+ and BH+.The other known nucleosides without standards were preliminary identified by MH+/BH+ matching.(2)Potentially new nucleoside were screened out by MH+/BH+matching.(3)some nucleosides that had protonated bases but without related base-ribose conjugate were discovered by MH+/BH+matching.Several substituents in 5’-OH of nucleosides in our in-house database were used for molecular prediction of nucleosides.The molecular formula,protonated molecule and protonated base accurate mass to charge ratio of predicted nucleosides were also calculated.Potentially new nucleosides were screened out by MH+/BH+matching.4.The structure confirmation of urinary and serum nucleosides by UHPLC Q-Trap/MSUHPLC Q-Trap/MS analysis was conducted by multiple reaction monitoring-information-dependent acquisition-enhanced product ion scan(MRM-IDA-EPI)mode to detect known nucleosides and potentially new nucleosides.MH+and BH+were selected as parent ion(Q1)and product ion(Q3)in MRM-IDA-EPI mode.Declustering potential(DP)and collision energy(CE)were optimized to obtain better sensitivity.Finally,a total of 67 nucleosides,including 20 nucleosides found from urine for the first time and 14 new nucleosides,were identified for urine.A total of 27 nucleosides were identified from serum.And there were 6 new nucleosides found from serum.5.The methodological study for comprehensive characterization of nucleosides by UHPLC Q-Trap/MSLinear range,limit of detection,repeatability,stability and precision were researched by UHPLC Q-Trap/MS.In conclusion,the systematic identification of urinary and serum nucleosides was satisfactorily fulfilled by combing UPLC Q-TOF/MS and UHPLC Q-Trap/MS in this paper.Thirteen new nucleosides were identified from urine and 6 new nucleosides were identified from serum firstly.The MH+/BH+matching method and molecular prediction strategy were effective tools for discovery of new nucleosides.This method developed in this study is beneficial for clinical research of urinary and serum nucleosides as cancer biomarkers.