| Thiol-containing compounds are widespread in the biological samples, and play important roles in redox balance and resistance to oxidative stress. Therefore, detection of thiol-containing compounds with high sensitivity and accuracy is important in biological research and disease diagnosis. However, only targeted analysis of several thiols was generally performed in the previous methods, and non-targeted profiling of thiols in the biological samples was rarely reported. MS is one of the most prominent platforms for metabolites detection due to its high sensitivity and specificity. However, certain compounds are absence of ionizable functional groups or their isotope standards are not commercially available, which will lead to the lower detection sensitivity and compromise the accuracy of the quantitation.Recently, stable isotope labeling (IL) strategy has been developed for non-targeted qualitative or quantitative profiling of metabolites with the MS-based platform. The typical IL method introduced a light and heavy isotope tags to the samples, respectively, then mixed the two labeled samples, followed by LC-MS analysis. It has been applied in metabolomics for the identification or quantification by calculating the peak intensity ratios of the isotope labeled peak pairs in two comparative samples. However, high-resolution mass spectrometer (i.e., FTICR or Orbitrap), which was high cost, applied in the IL-LC-MS method, and the MS was normally operated in full scan mode. If using low-resolution instrument, the detection will face the problem of lower sensitivity, selectivity and accuracy.In our text, we used the IL strategy in combination with common triple quadrupole mass spectrometer under different scan mode for analysis of thiols. The developed method can significantly improve the identification sensitivity, selectivity and accuracy, then, it can solve the aforementioned problems and provide novel ideas for qualitative and quantitative profiling of metabolites with the MS-based platform. The research contents were as follows:1. Four kinds of derivatization reagent and their derivatized thiols were evaluated. Compared with underivatized thiols, the fragment ions of derivatized thiols were mainly generated from the derivatization reagent moiety, and more likely to produce the same ions. We investigated the detection sensitivity of derivatized thiols by using RPLC-MS both in the SIM and MRM mode. Our results show that hydrophobic group and quaternary ammonium salt group have positive effects on mass response. In addition, we also found that the MS fragmentation behavior was releated with the MRM sensitivity, and the MRM signal will be higher when the fragmentation behavior was simple. We selected the ?-bromoacetonylquinolinium bromide (BQB), which was containing quaternary ammonium, as derivatization reagent in the following experiments.2. A highly sensitive method was developed for the detection of phytochelatins (PCs) in rice by stable isotope labeling coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (IL-LC-ESI-MS/MS) analysis. A pair of isotopes labeling reagents (BQB and BQB-d7) were used to label PCs in plant sample and standard PCs, respectively, and then combined prior to LC/MS analysis. The heavy labeled standards were used as the internal standards for quantitation to minimize the matrix and ion suppression effects in MS analysis. In addition, the ionization efficiency of PCs was greatly enhanced by 14-750 folds and the m/z of PCs was decreased around m/z 428-453 through the introduction of a permanent charged moiety of quaternary ammonium of BQB into PCs. Furthermore, under cadmium (Cd) stress, PC2?PC3?PC4, and PC5 were detected in the rice by this method, and the contents of PCs in rice dramatically increased with the increased concentrations and treatment time of Cd.3. Here we developed a novel strategy of isotope labeling in combination with high performance liquid chromatography-double precursor ion scan-mass spectrometry (IL-LC-DPI-MS) analysis for non-targeted profiling of thiol-containing compounds in beer. In this strategy, a pair of isotopes labeling reagents, BQB and BQB-dy, were used to selectively label thiol-containing compounds in beer. Then BQB and BQB-d7 labeled sample solutions were mixed (1/1, v/v) and detected by LC-DPI-MS. The BQB and BQB-d7 labeled compounds can generate two characteristic product ions m/z 218 and 225, which contain isotope tag and therefore were used for double precursor ion scans in mass spectrometry analysis. Extracted characteristic peak pairs from the two precursor ion scan spectra and the peak pairs with the same retention times and intensities were assigned as candidates. Using the strategy,21 thiol candidates were discovered in beer. It is worth noting that Nac existence in beer was reported by our method.4. Precursor ion scan and multiple reaction monitoring scan (MRM) are two typical scan modes in mass spectrometry analysis. Here, we developed a strategy by combining stable isotope labeling (IL) with liquid chromatography-mass spectrometry (LC-MS) under double precursor ion scan (DPI) and MRM for non-targeted profiling and relative quantitation of thiols from 5 types of cancers and healthy controls. Firstly, the IL-LC-DPI-MS method was applied for non-targeted profiling of thiols, and 103 thiol candidates were discovered in urines. It is worth noting that pantetheine was identified in human urine. Secondly, the IL-LC-MRM-MS method was developed for relative quantification of thiols in cancers compared to healthy controls. In MRM mode, all the precursor ions of [M]+and [M+7]+for the MRM transitions of [M]+2?218.1 and [M+7]+?225.1 were acquired from the BQB and BQB-d7 labeled samples by using aforementioned DPI method. Compared to IL-LC-DPI-MS method, the sensitivity of IL-LC-MRM-MS improved by 2.1-11.3 folds. Our study presents the report for the increased level of HCys and ?-GluCys in urine of nasopharyngeal cancer and gastric cancer, respectively, and pantetheine was significant decrease in both esophagus and lung cancer. |
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