Analyses Of Small Biomolecule Glutathione By Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Matrix-assisted Laserdesorption Ionization time-of-flight Mass Spectrometry(MALDI-TOF MS)has been developed and improved for more than 30 years since its advent in the 1980 s.It has many advantages such as high sensitivity,high throughput,soft ionization,good salt tolerance,simple sample preparation,fast analysis speed and less sample consumption,which makes it easier for people to obtain much complete samples information.Therefore,it has been vigorously developed and widely used in the detection of biological samples.And it has incomparable advantages over other analytical methods in the analysis of polypeptide,protein,biological metabolites and microorganisms.I practical application,mostly,MALDI-TOF MS needs the use of matrix to assist for the analyte ionization.The commonly used matrices are usually small organic compounds,they are produced many matrix signal peaks in the lower mass to charge areas(<600 Da),seriously interfering the small molecules to be measured,and unenven cocrystallization of analyte and matrix makes the reproducibility of detection results poor.Therefore,it is challenging to apply MALDITOF MS for the qualitative and quantitative analysis of small molecules..To develop the MALDI MS method for the dection of-small molecules,glutathione(GSH),a small molecule polypeptide with high biological significance,was selected as our target analyte.The qualitative and quantitative analysis of endogenous GSH in cancer cells was successfully achieved by developing derivative reagents and employing appropriate internal standard.There are three chapters in this paper.The specific contents are as follows:The first chapter is the introduction.In this chapter,the principle of MALDI-TOF MS and the models of its ionization mechanism are introduced and discussed in detail.The requirements and types of matrices,sample preparation methods,pretreatment methods and the shortcomings of MALDI mass spectrometry were also discussed.Finally,targeting to the shortcomings,the solutions and the research plans are put forward.In the Chapter 2,we developed a strategy for labeling GSH efficiently by using quinoline-5,8-dione(QLD)with aromatic ring structure and strong ultraviolet absorption.The method was applied to the accurate analysis of glutathione(GSH)via MALDI-TOF MS.By series of experiments,we optimized the reaction time,temperature,MALDI matrix,detection methods and additives.The detection limit of glutathione using our method can reach 10 fmol/μL.At the same time,we designed an ECA with only one methyl difference from GSH as an internal standard,which can assist for accurate measuring the concentration of GSH in the range of 4-4000 mM by using the relative intensity ratio of QLD-GSH to QLD-ECA.On this basis,we further successfully detected the content of endogenous GSH in HeLa cell fragmentation products.Our results are consistent with those of the commercial ultraviolet colorimetric kit,which further illustrates the reliability of our method.This method is fast,low cost,rapid detection and high sensitivity.It can provide a new tool for the determination of glutathione.In Chapter 3,based on the previous experiment,we designed a label-free MALDITOF MS analysis strategy for small biomolecules by using traditional matrix CHCA and GSH for one-step rapid and efficient derivatization.In this method,CHCA was used as a reactive matrix for the specific analysis of GSH.That is to say,it overcomes the interference of small molecular matrix on the detection results,and avoids the unknown influence of exogenous tags on the detection results.Finally,we compare this method with the traditional direct detection method,and verify the prospects of this method in practical application in the future.