Investigation Of In Situ Derivatization Mass Spectrometry And Its Application In Single Cell Analysis

Cellular heterogeneity is a widespread phenomenon.In conventional cell population analysis(mean results of a large number of cell analyses),the averaging process will mask the characteristic information of individual cells and miss the heterogeneous results between cells.Cellular heterogeneity information might play a vital role in early diagnosis and treatment of cancer.Single cell analysis provides new insight to cellular heterogeneity at genomics,transcriptomics,proteomics,and metabolomics levels,and also provides an opportunity to further the current understanding of biological variability and differential response to disease and therapeutics.In particular,small molecule metabolites,as an important part of the intracellular environment,directly reflect cell responses and cell phenotypes.They are critical players in many life processes,such as energy balance,intercellular communication,and osmotic regulation.However,due to small volume(pL),extreme variation of metabolites types and levels,wide spans of concentration,and lack of amplifying technologies of single cell,there are still a lot of metabolites that could not be detected at single cell level.This puts big challenges and high demands on sensitivity of single cell analysis methods.In recent years,in situ derivatization reactions have developed rapidly.The introduction of charge tags through various derivatization reactions to enhance the mass spectrometry(MS)response of target analysis is widely used in the in situ analysis of chemical substances in biological samples,such as serum/plasma and tissues.However,achieving in situ derivatization at the single cell level is still very challenging,since it is necessary to meet the both conditions of in situ derivatization and cell compatibility.Large cell disturbances or changes in cell state will cause changes in the types and concentration levels of metabolites in the living cells.However,the current derivatization methods mostly use organic reagents,UV light and other critical conditions,which limit its application at single living cell.Therefore,it is urgent to develop a new in situ derivatization reaction and apply it at the single cell to enhance the sensitivity of metabolites in mass spectrometry analysis.We developed a variety of new in situ derivatization reactions,which respectively achieved in situ N-nitrosamine degradation,in situ derivatization and analysis of total homocysteine(tHcy)in serum and urine samples.Finally,the cell compatible click reaction was successfully applied to in situ derivatization in living cells.This paper performed from the following three aspects:1.In situ degradation of N-nitrosamine via corona discharge at ambient pressure.The highly reactive free electron is produced via corona discharge and responsible for in situ degradation of three N-nitrosamines to form the corresponding secondary amines as the degradation products.To further explore the mechanism of the highly selective degradation of N-nitrosamines,we designed a series of experiments to investigate whether the degradation reaction was closely related to corona discharge at ambient pressure.This investigation was carried out from corona discharge-related experiment conditions,such as spray voltage,nebulizer gas,distance between spray emitter to MS inlet,and solvent composition.Then,we applied an electric filed to substances generated in corona discharge to further prove that the degradation is related to the negatively charged species produced from corona discharge.Finally,we then conducted theoretical calculations to confirm electrons have been involved in these degradation reactions.2.Click reaction was used to detect total homocysteine(tHcy)and related metabolites in serum and urine samples.For this study,we designed the 2-cyanobenzothiazole based,charge tag-containing probe,as[(2-Cyano-benzothiazol-6-ylcarbamoyl)-methyl]-trimethyl-ammonium(NCBT),which could selectively couple to analytes containing 1,2-aminothiol group or 1,3-aminothiol via click reaction for adding charge tag(permanent charged quaternary ammonium group)to enhance MS response.Elevated plasma tHcy has been recognized as independent risk factor for a variety of vascular diseases.We used charge tag labeled probes to specifically and rapidly derivatize Hcy in serum and urine to enhance the performance of Hcy.To accomplish rapid MS measurement of tHcy,we also introduced induced nanoelectrospray(InESI),as a modified nESI,which has the ability to provide a stable MS signal and alleviate severe ion suppression effects in the biological fluid samples with excess derivatization reagent.Moreover,we applied this method to quantitation of tHcy in bovine serum and urine with good reproducibility and recovery.In addition to Hcy,other metabolites contained or related in Hcy metabolic pathway also could be simultaneous detection.3.Under the premise of enhanced performance of Hcy in complex biological fluids via in situ derivatization with NCBT,we tried to apply the synthesized charge tag containing probes NCBT to increase MS response in single living cells.To achieve this goal,we first demonstrated that the NCBT-Cys click reaction having highest reaction efficiency under physiological conditions(pH=7.4).Second,the cells have good viability after incubation with NCBT,suggesting NCBT owns good biological compatibility for single living cell experiments.Compared to Cys without charge tag,MS signal intensity of NCBT-Cys could be improved up to～75-folds in aqueous solutions due to the fact that permanent charged ion would dominate the charge competition during ionization process.Therefore,this new method offers an opportunity to achieve the monitoring of Cys concentration and its dynamic alterations of Cys concentration after drug stimulation in single living cell.Although Cys was demonstrated as proof of principle,this strategy should be readily extended to many other metabolites.With all the efforts to push forward SCMS metabolites,we believe that much wider applications could be anticipated for clinical diagnosis,understanding disease pathogenesis and therapeutic drug discovery.