The Application Of Photoionization Mass Spectrometry In Biological Tissue Imaging And Tea Thermal Processing

Mass spectrometry(MS) is an analytical method based on the mass-to-charge(m/z)of ionzed species for the chemical analysis.It has become one standard method for qualitative and quantitative analysis and has been applied in medicine,life sciences,environment,medicine and environment for its advantages such as high sensitivity,high accuracy,high specificity,high throughput and fast analysis speed.In-situ imaging analysis of polar and nonpolar compounds on the surface of biological tissues and real-time online analysis of various gaseous compounds during the tea thermal processing are one of the most important challenges in the field of mass spectrometry.At present,the improvement and innovation of ionization methods are an important trend to solve the above-mentioned challenges.Photoionization technique has many advantages,such as "soft ionization" characteristic,no ionization polarity discrimination,high sensitivity and strong anti-matrix effect,and has been gradually applied to the direct analysis of many compounds in complex matrix samples.Desorption electrospray ionization(DESI)-MSI is an in-situ MSI technique working in an open environment.It can quickly image small molecular metabolites and lipids through direct scanning of tissues with high throughput.However,the limitation of DESI-MSI is the lower sensitivity for the detection of neutral and non-polar compounds because of the complex matrix environment and polar discrimination phenomenon of ionization in biological tissues.The detection of polar and non-polar volatile compounds in tea thermal processing is mainly based on chromatography-mass spectrometry tandem technique.The ion source used is usually electron ionization.The chromatographic separation time of this method is long,and it is impossible to carry out real-time online monitoring.In this paper,we propose two photoionization mass spectrometry techniques for the direct analysis of polar and non-polar volatile compounds in biological tissue imaging and tea thermal processing.The first chapter of the thesis mainly introduces the research overview of mass spectrometry imaging technique,including the working principle,characteristics,advantages and disadvantages,classification,and application.Among them,three mainstream mass spectrometry-imaging technologies are introduced in detail.This chapter also introduces the principles and characteristics of photoionization and the application of photoionization mass spectrometry technique in biology.The second chapter of the thesis introduces the methodology of desorption electrospray ionization/postphotoionization mass spectrometry imaging(DESI/PI MSI).The method mainly combines desorption electrospray ionization with an atmospheric pressure postphotoionization interface.DESI/PI mainly includes a transfer tube,a vacuum ultraviolet discharge lamp,an ionization tube,and an airflow assisted transport arrangement.First,DESI and DESI/PI techniques are used to detect the standards with different polarities,respectively.Secondly,the types and proportions of spray solvents are optimized to improve the sensitivity of DESI and DESI/PI.Finally,the influence of the opening/closing of transport arrangement on the signal intensity of compounds are explored.The experimental results show that a large number of neutral small molecular metabolites and weak polar and non-polar cholesterol,creatine,glutamine,and GalCer lipids in the mouse brain could be detected and imaged by DESI/PI.Besides,the neutral polar catechins in the tea leaf tissue have high sensitivity in the DESI/PI mode,and PI can also increase the signal intensity of polar caffeine by 200 times.The third chapter introduces the study of melanocytic nevi by desorption electrospray ionization/photoionization mass spectrometry imaging.Melanocyte nevi is the commonest benign tumor in humans and has a certain risk of developing malignant melanoma.Currently,clinical and pathological analysis are still the gold standard to diagnosis the melanocytic nevi.However,this process is relatively tedious and time-consuming,and the diagnosis result depends largely on the skills and experience of surgeons,which can lead to some perceived subjectivity.To provide a molecular method for detecting melanocytic nevi and to find potential biomarkers positive-related to melanocytic nevi,a comprehensive and systematic visual analysis of endogenous compounds in melanocytic nevi is particularly important.The experimental results show that by using these two complementary imaging techniques,as many as 118 compounds in the sample tissue were detected,and it was found that some specific lipids can be used to distinguish the nevi region from normal tissues,such as cholesterol,S1P,PE,FAs,MAG,and DAG,etc.Multivariate statistical analysis(box plot,T-Test,and PCA)were used to find compounds that are different in nevi and normal tissues and we found that cholesterol played an important role in distinguishing the two tissues.Immunohistochemistry(IHC)experiments further verified the reason for the accumulation of cholesterol in melanocytic nevi from the protein level.The fourth chapter introduces the study of gas-phase intermediates and volatile compounds released from thermal process of tea by synchrotron radiation photoionization mass spectrometry.First,at a fixed temperature,the volatile compounds released during the baking process were preliminarily characterized by adjusting the photon energy,besides,the headspace GC/MS experiment was used to assist the qualitative.Then the effects of different baking temperatures,atmosphere,and time on the formation of water,gas-phase intermediates,and volatile compounds released during the roasting process are respectively explored.The experimental results show that the first process that occurs in tea roasting is the dehydration process.Subsequently,various gaseous aromatic compounds are released through the Maillard reaction and oxidative degradation of lipids.Studies have found that oxygen in the air can accelerate the formation of a variety of volatile compounds,and has a great influence on the formation of 5-hydroxymethylfurfural(5-HMF),an intermediate in the Maillard reaction.The three sulfur-containing compounds,dimethyl sulfide,hydrogen sulfide,and methanethiol produced in the baking of green tea are gradually generated,and the possible mechanisms are discussed.Finally,the principal component analysis verifies that the time-resolved spectrum can be used to predict the degree of tea roasting,and roughly divides the roasting process into four stages.Finally,this thesis summarizes the innovation points in the work,and objectively discusses the deficiencies in the work and the directions that can be improved in the future.(1)Although the DESI/PI-MSI technique currently established has high sensitivity,the imaging resolution is low,nano-DESI or laser can be chosen to futher improve the resolution and sensitivity instead of the DESI desorption source.(2)A synchrotron radiation source or laser can be used to replace the VUV lamp,because the photon density and brightness of VUV are not high enough,and the photon energy is not adjustable.(3)The current DESI/PI-MSI technique has a narrow application field,and it can be applied to the visual analysis of medicines,plants,food,traditional Chinese medicine,and more diseased tissues.(4)The current DESI/PI-MSI technique can only perform qualitative and relative quantitative analysis of the analyte in the sample,and it is necessary to establish a complete set of quantitative methods.(5)The mass spectrometer used in the current mass spectrometry imaging experiment is a time-of-flight mass spectrometer,which cannot accurately identify the compounds by MS/MS and can be equipped with a mass spectrometer with a second mass analyzer.