Simultaneous Detection Of Bioactive Small Thiol Molecules In Biological Samples By Paper Spray Mass Spectrometry

Bioactive small molecules,such as bioactive peptides,bioactive amino acids and derivatives,amines,lipid media,and gaseous signal molecules,play an important role in maintaining the steady state of human organ structure and function.This kind of molecules has the common characteristics of small molecular weight,simple structure,wide tissue distribution,and various biological effects.Their function and level have important pathological significance.Among them,biothiols is one of the most important bioactive small molecules,which play an essential role in various biological metabolic processes,including oxidative stress,protein modification,signal transduction,apoptosis and cell differentiation.More than 100 biothiols have been identified in biological fluids,among which cysteine?Cys?,homocysteine?Hcy?,glutathione?GSH?,and N-acetylcysteine?Nac?Cysteinyl glycine?CysGly?are the main thiols in the transsulfur pathway.Based on the important physiological functions of thiol compounds,monitoring the levels of Cys,Hcy,GSH,Nac,and CysGly in biological fluids is necessary for early diagnosis of disease and assessment of disease progression.Although a variety of detection methods have been developed at present,they are basically derivatization methods,and most of them have problems such as long analysis time,high cost,and complicated operation.Paper spray ionization,as a new type of ambient ionization technology,not only has the advantages of ambient ionization,but also is easy to operate and does not require tedious pretreatment.As a method to replace traditional sample preparation and chromatography,paper spray mass spectrometry has proven to be able to ionize various molecules in complex biological samples with significant sensitivity.In view of the existing problems,it is necessary to develop a method based on paper spray mass spectrometry for the detection of biothiols for rapid and accurate analysis of biothiols.Using the designed and synthesized Ebselen-NH₂ probe containing the"selenium tag",we developed a paper spray ionization mass spectrometry method for rapid derivation of biothiols on paper substrates.Combined with the selective reaction monitor?SRM?mode of the ion trap mass spectrometer,a high sensitivity,high selectivity,and high throughput analysis of the biothiol content in biological samples was achieved.By selecting representative samples,such as blood,cell lysate,etc.,the biothiol content can be qualitatively detected and accurately quantified,and the results can provide theoretical basis for the diagnosis and treatment of clinical diseases.This thesis mainly includes the following:A biothiols detection method combining chemical derivatization and paper spray ionization was proposed.In this method,a probe Ebselen-NH₂ was designed and synthesized.The probe contains a"selenium tag",and has a specific response to biothiols and can form Schiff bases through the interaction of aldehyde groups and amino groups,which can be covalently fixed on the surface of oxidized paper substrate.First,we treated the paper substrate to oxidize from the surface hydroxyl groups?-OH?to aldehyde groups?-CHO?,and verified the oxidation using2,4-DNP.Then we explored the pattern of probe immobilization,and investigated the conditions of the amount of probe and incubation time.After that we investigated the influencing factors affecting the ionization performance of paper spray,such as the applied voltage,the distance between the paper substrate tip and the MS inlet,the angle of the paper substrate tip,and the type of solvent,and obtained the optimal experimental conditions.Furthermore,the derivatization effect of biothiols on paper was demonstrated under these conditions.Through the SRM mode of the mass spectrometer,the qualitative and quantitative analysis of five biothiols were completed,and the biothiols in plasma samples and cell lysates were determined.The experimental results showed that the method had good linearity in a wide range in different matrices,and the limit of detection was very low,which can be as low as 0.06 nmol/mL.This method is expected to be used for the detection of other bioactive small molecules,which will help to reveal and understand the biological and pathological role played by such bioactive small molecules,and provide a theoretical basis for related research.