Study On Small Biological Thiol Molecules By Matrix Assisted Laser Desorption Ionization Mass Spectrometry

Matrix-assisted laser desorption ionization mass spectrometry(MALDI-MS)is characterized by ease of operation,high throughput,high sensitivity,high salt tolerance,and single electron ion peaks,and is therefore widely used in the fields of proteomics,microbial identification,tissue imaging,and biomarker screening.In general,matrices are required to assist the ionization of analytes during MALDI-MS detection.However,conventional matrices are usually organic small molecules that produce strong matrix background signal interference in the low molecular weight region of the mass spectrum,resulting in a great challenge for the analysis and detection of small molecule compounds(?600 Da)by MALDI-MS.Small biological thiol molecules,such as cysteine(Cys),homocysteine(Hcy),reduced glutathione(GSH)and oxidized glutathione(GSSG),are a class of important compounds in living organisms.They are responsible for the regulation of redox balance in living organisms and act as potential biomarkers in many diseases.Therefore,derivatization of small biological thiol molecules is often required for their determination.Currently,a commonly used detection method is fluorescence spectrometry.However,this method is often difficult to accurately detect Cys and Hcy due to their extremely similar structures,besides,the insufficient analytical throughput also limits its application.Notably,MALDI-MS has high detection throughput and can achieve accurate determination according to the molecular weight.However,it has limitations in the detection of small molecule compounds.To address this issue,here we aimed at developing MALDI-MS labels for the selective detection of thiol molecules.These labels can,on the one hand,selectively increase the molecular weight of the analytes to keep them away from low molecular II weight regions and free from severe interference of matrix background signals.On the other hand,the labeling can improve the ionization efficiency,thus enabling simple,rapid and high-throughput MALDI-MS-based detection of biological small thiol molecules.The details of the study are as follows:?.The traditional MALDI-MS matrix CHCA can react with GSH containing free sulfhydryl groups.After optimization,the detection sensitivity of GSH can be improved in the positive ion mode of MALDI-MS.Experimental results show that when CHCA is the normal matrix concentration,it can be derivatized with GSH in a molar ratio of1:1,and the remaining CHCA can continue to be used as matrix for ionization.Therefore,CHCA can be a reactive matrix for the detection of GSH.In addition,we also evaluated the reproducibility of the derivatized mass spectrum signal.The diffracted samples have high reproducibility.Due to the combination of CHCA and GSH,the ionization efficiency of GSH is improved,and the limit of detection of GSH is reduced by two orders of magnitude compared with that without derivatization.This method is also effective for other compounds containing free sulfhydryl groups.Finally,we applied this method to the detection of GSH in HepG2 cells.This method can effectively resist the interference of complex systems and successfully detect GSH in the intracellular environment in the positive ion mode of MALDI-MS.?.Previously,we developed a method of selectively labeling GSH with quinoline-5,8-dione(QLD)and using glutamate-cysteine-alanine(ECA)as internal standard.to quantify the GSH in He La cells.Herein,we further studied and developed a rapid and reproducible quantitative analysis method for the detection of GSH and GSSG in red wine based on MALDI-MS.This is the first application of MALDI-MS in the quantitative detection of GSH and GSSG in wine.According to our previous work,the concentration of free GSH in red wine samples was first determined.Then,Glutathione Reductase(GR)was used to reduce red wine samples,and the label QLD and internal standard ECA were added to the same sample in turn,so as to realize the quantitative analysis of GSH and GSSG in red wine samples by MALDI-MS.Using this method,the quantitative analysis of GSH and GSSG of four different brands of commercial red wine exposed to air for seven days was realized.In addition,the method was further extended to detect GR enzyme activity with GSSG as a probe.The determination of GR enzyme activity in human serum samples was realized.?.To achieve simultaneous quantitative detection of Cys and Hcy by MALDI-MS,we developed 1-Pyrenecarboxaldehyde(1-py)label that can specifically recognizes Cys and Hcy based on the cyclization reaction of aldehyde group with sulfhydryl and amino groups and quantitatively detect them with high sensitivity and selectivity.1-py is highly ionized in MALDI-MS due to its biphenyl structure.Its combination with Cys and Hcy allows the ionization efficiencies of the analytes to be improved.After derivatization,the limit of detection of Cys and Hcy can reach amol level with good reproducibility.Due to the excessive addition of 1-py in the derivatization process,when the concentration of the substance to be measured changes,the detection signal strength of the remaining labels after the reaction also changes.Therefore,within a certain concentration range,there is a linear relationship between the proportion of the detection signal strength of derivative products in the total signal strength of products and remaining labels and the concentration of the substance to be tested.Thus,the quantitative detection without internal standard was achieved.Finally,1-py was successfully used for simultaneous quantitative analysis of Cys and Hcy in HepG2 cells.In summary,by using 1-py as a label,we have realized a simple,sensitive and MALDIMS-based method for quantitative analysis of Cys and Hcy without internal standard.