Liquid Chromatography-mass Spectrometry For The Study Of Modified Nucleosides

RNA,occupying a central position in the central dogma,who gets genetic information from DNA and pass it to the protein,plays a decisive role in the process of physiological activities In all organisms.Various chemical modifications of RNA have been found as early as half a century ago,and to date the type is over one hundred kinds.In 2010,scientists found FTO,s activity on m6A,which is the first time that people know RNA methylation was reversible.Post-transcriptional RNA modifications can be dynamic and might have functions beyond fine-tuning the structure and function of RNA.Understanding of these RNA modification pathways and their functions may allow researchers to identify new layers of gene regulation at the RNA level.However,the effect and function of RNA modifications are still poorly understood.The present relevant research techniques are imperfect.For example,the sensitivity of detection method is not high enough to carry out research on the rare samples or to accurately quantify modified nucleosides of low content.There is an urgent need to develop new methods for in-depth analysis of RNA modifications,including accurate quantification and localization.Owing to the high sensitivity and selectivity of liquid chromatography coupled with mass spectrometry(LC-MS/MS),in this study,we established some sample pretreatment techniques combined with LC-MS/MS to further improve the sensitivity of detection to achieve qualitative and quantitative analysis of RNA modified nucleosides.Meanwhile,selective enzyme cleavage technique combined with LC-MS/MS was established to study the position and physiological function of N7-methylguanosine.1.We developed a DSPE-LC-MRM-MS/MS method for comprehensive profiling of ribose conjugates in human follicular fluid.Ribose conjugates can be readily purified using CeO2-based DSPE strategy.Using this method,50 potential ribose conjugates were identified in human follicular fluid.The follicular fluid from healthy controls and polycystic ovarian syndrome(PCOS)patients can be clearly differentiated with the partial least squaresdiscriminate analysis based on the detected ribose conjugates.In addition,8 ribose conjugates exhibited significant differences between PCOS patients and healthy controls,which could potentially serve as the indicator of PCOS.2.We developed a DSPE-SIL-LC-DNLS-MS method for comprehensive profiling of ribose conjugates in human urine.Ribose conjugates can be readily purified using CeO2-DSPE strategy due to its high affinity and specificity,which can efficiently remove the complex matrix of biological samples.The enriched ribose conjugates were labeled with acetone and acetone-d6 followed by double neutral loss scan-mass spectrometry analysis,which can significantly facilitate the identification of ribose conjugates.Using this method,49 ribose conjugates candidates were successfully identified in human urine.Moreover,the contents of 7 ribose conjugates were found significantly different between healthy controls and lymphoma patients,which could potentially serve as the indicator of lymphoma.3.We developed a nucleic acid enzyme digestion technology combined with liquid chromatography-tandem mass spectrometry method to investigate the N7-methylguanosine(m7G)modification status in intetrnal mRNA.Using this method and taking the advantage of the difference between S1 nuclease and phosphodiesterase I,m7G was detected in internal mRNA of higher eukaryotes including plants,human cells and rat tissues,with the contents varying in different cell types.Moreover,we found that Cadmium stress can change the contents of m7G in plant RNA,suggesting the functional roles of m7G in response to environmental stresses.In all,our results and findings set the basis for further understanding of the biological functions of RNA modifications.