Chemical Tagging Assisted Liquid Chromatography-mass Spectrometry For Analysis Of Nucleotides

Nucleotides are consisied of phosphate group,sugar ring and base.Apart from being the building blocks for nucleic acid,nucleotides are also involved in many important biological processes.Nucleosides analogues and their related phosphorylated metabolites play critical roles in tumor metabolism.Nucleotides analysis can provide valuable information of diseases and some proble ideas for treatment of diseaes.However,determination of the endogenous nucleotides from complex biological matrix is still challenging due to their high structural similarity and high polarity.Some methods have been established to detect cellular nucleotides.Capillary electrophoresis(CE),and high-performance liquid chromatography(HPLC)with UV detection were applied to detect ribonucleotides.But these methods generally have low detection sensitivities and weak capability on identification for ribonucleotides.Liquid chromatography mass spectrometry(LC–MS)has been frequently employed for analysis of nucleotides.Nucleotides have weak retention on the reversed-phase column due to their high priority,which could lead to the poor separation of ribonucleotides.To improve the retention of ribonucleotides on reversed-phase chromatographic separation,ion-pairing reagents were usually added to mobile phases.But these additive reagents could contaminate mass spectrometer and induce ion suppression to analytes,which eventually cause the low detection sensitivities of analytes.Hydrophilic interaction chromatography(HILIC)coupled with MS is an alternative technique for detection of nucleotides,but peak tailing and drifting are frequently observed for separation of nucleotides,which would lead to the poor reproducibility as well as low detection sensitivities.CE–MS-based approaches also have been established to sensitively detect polar phosphorylated metabolites,but the lab-fabricated capillaries or emitters are frequently needed.In this study,(1)A pair of light and heavy stable isotope labeling reagents,2-(diazomethyl)-N-methyl-N-phenyl-benzamide(2-DMBA)and d₅-2-(diazomethyl)-N-methyl-N-phenyl-benzamide(d₅-2-DMBA),were synthesized to label ribonucleotides.2-DMBA showed high specificity and high efficiency for the labeling of ribonucleotides.Our results demonstrated that the detection sensitivities of 12 ribonucleotides increased by17-172 folds upon 2-DMBA labeling.The obtained limits of detection(LODs)of ribonucleotides ranged from 0.1 fmol to 0.4 fmol.Using this method,we achieved the sensitive and accurate detection of ribonucleotides from only a few cells(8 cells).In addition,we found that the contents of almost all of these ribonucleotides were significantly increased in human breast carcinoma tissues compared to tumor-adjacent normal tissues,suggesting that endogenous ribonucleotides may play certain functional roles in the regulation of cancer development and formation.Taken together,the diazo reagent labeling strategy with mass spectrometry analysis enables highly sensitive detection of ribonucleotides.(2)We synthesized a paired reagents of light and heavy isotopemers,2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone(DMPI)and d₃-(2-(diazomethyl)phenyl)(9-methyl-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)methanone(d₃-DMPI).The paired reagents of DMPI and d₃-DMPI carry the diazo group that can efficiently and selectively react with the phosphate group on polar phosphorylated metabolites.The developed DMPI/d₃-DMPI tagging with LC–MS analysis enabled the sensitive determination of 12 kinds of ribonucleotides from a single cell and from a single stamen of Arabidopsis thaliana(A.thaliana).Moreover,the quantification results demonstrated that the levels of endogenous ribonucleotides dramatically decreased upon stress by high temperature.Therefore,we developed methods with high sensitivities for ananlysis of nucleotides.And 12 nucleotides in a single He La cell can be detected,demonstrating the methods exhibit advantags for determination of nucleotides in rare cells,such as circulating tumor cell.The methods also present great potential for analysis of phosphorylated compounds.