Nano Matrix-Assisted MALDI Mass Spectrometry For Applications Of Detecting Small Molecules And Pharmaceutical Research

Matrix assisted laser desorption/ionization(MALDI)mass spectrometry(MS),as a"soft" ionization technology,has been widely used in biomedical field and promoted the research onnucleic acids,proteins and peptides.However,the traditional organic matrix is subject to the shortcomings of high background noise(a large number of matrix fragments detectable at m/z<1000)and low signal reproducibility caused by irregular crystallization(micron sized crystals),thus limiting the applications of MALDI MS in small molecular analysis.There is an urgent need to develop new MALDI matrices.In my thesis,I have developed nano matrices based on carbon or silicon nanomaterials for MALDI MS,and demonstrated a series of biomedical applications including identification and detection of small molecular substances,drug screening,and mechanistic revelation of the photothermal treatment of tumors with nano materials.My thesis is composed of the following sections:Chapter 1:a brief overview of MALDI MS technology,the development,applications and limitations of various matrices.Applications of MALDI MS in various fields,including high-throughput screening,new drug research and development,etc.The rationales of my PhD projects are also briefly introduced in this chapter.Chapter 2:It is necessary to develop rapid and sensitive methods of monitoring antibiotics in trace amounts in order to prevent their abuse.In this chapter,I have developed graphite dots(GDs)as a new MALDI matrix to identify small molecular antibiotics and their metabolites,and monitor their absorption and uptake,blood drug concentrations,organ distribution and chemical structure changes in vivo,and demonstrated highly sensitive quantification(the limit of detection in the range of femtomole);Combining this technique and histomorphological characteristics of living organ sections,the metabolic changes of antibiotics can be visualized in great details.Chapter 3:In this chapter,I have developed an integrated drug screening and pharmacokinetic platform(GLMSD)by taking advantage of computer-aided highthroughput drug virtual screening and graphite dots-assisted laser desorption/ionization mass spectrometry.The platform has been applied to quickly screen the library containing 240000 compounds,allowing for successful identification ofnew compounds as hexokinase 2(HK2)inhibitors for glioblastoma chemotherapies.In comparison to the traditional drug screening methods,GLMSD has the following advantages:(1)Label-free,no limitation of so-called "blind zones" in screening;(2)High sensitivity,improved resistance to salts,simplified sample pretreatment;(3)Higher throughput performance than traditional LC-MS screening by 12 times;(4)Minimum sample volume(10 μL per test),and making the least consumed experimental animal possible for "one mouse completes a complete pharmacokinetic curve".The representative compound 27 identified by our screening can effectively inhibit the activity of HK2 enzyme in tumor cells.Our experiments reveal the mechanisms of compound 27 normalizing the reprogramming glycolytic pathway of glioblastoma cells.In the subcutaneous tumor and in situ brain tumor mouse model,it has been demonstrated that HK2 inhibitor(compound 27)and temozolomide can synergistically kill glioblastoma and achieve optimal therapeutic effect.The integrated platform of GLMSD is expected to be an important tool for rapid and efficient pharmaceutical research and development.Chapter 4:Nanoparticle mediated photothermal therapy(PTT)has entered to the early stage of clinical trials.A better understanding of the molecular mechanisms of PTT is very important to improve the therapeutic efficacy and clinical applications.In this chapter,I have investigated on the reprogramming molecular mechanisms of how nano materials improve tumor hypoxia in the photothermal therapy.Single layer titanium carbide@gold(Ti3C2@Au)has been synthesized as the photothermal agents to improve tumor hypoxia and specifically induce cancer cell apoptosis.The nanomaterials can also serve as the MALDI matrix to detect the key small molecules sin hypoxia intervention and apoptotic 4T1 cells.Metabolomic data allows me to deeply discuss the molecular signal pathways of PTT to alleviate tumor hypoxia.I have identified that phospholipase C plays a very important role in the regulation of tumor hypoxia and apoptosis.Photothermal treatment of nanomaterials improves the hypoxia of tumor cell by regulating key pathways such as energy metabolism and amino acid metabolism.Continuous photothermal therapy can induce tumor cell apoptosis.Chapter 5:In sports events and food safety inspection,monitoring of banned drugs and illegal additives calls for the development of sensitive and rapid methods.In this chapter,I have synthesis hybrid Ir/SiNW,as a new MALDI matrix,for the detection of small molecular illegal drug(clenbuterol).The nano matrix has the characteristics of sufficient UV absorption,low noise background and high efficiency in assisting small molecule ionization.The Ir/SiNW matrix has been demonstrated for sensitive detection of clenbuterol(LOD:0.18 pmol)and other small molecules with good reproducibility.In comparison to the individual components(Ir or SiNW nanomaterials),the as-prepared in situ hybrid Ir/SiNW matrix can increase the MS signal intensity by 10 times under the identical experimental conditions.After taking into account of hydrogen atom transfer and enhanced protonation at the interface of hybrid nanostructures,I am able to provide a better mechanism explanation for the high performance of this matrix.The hybrid Ir/SiNW assisted MALDI mass spectrometry can quantitatively detect clenbuterol in complex biological samples,offering a convenient and effective method for the monitoring of illegal additives.Chapter 6:The major results of my PhD projects is systematically summarized.I have tried to analyze the shortcomings of my current approaches,and provided an outlook of them in the future.In conclusion,my PhD thesis focuses on the development of carbon or silicon based nano-matrices and their biomedical applications.These nano matrices aim at addressing the problems of strong background noise of traditional MALDI matrices in the detection of small molecules.With these nano-matrices,I have performed a series demonstrations including highly sensitive detection of small molecule compounds,new drug screening against glioblastoma,and mechanistic revelation of photothermal treatment for tumor hypoxia improvement.These results are of great significance to promote the methodology of MALDI MS and broaden the applications of MALDI MS in biomedical research.