Stable Isotope Labeling Combined With Mass Spectrometry For Carbonyl Compounds Analysis

Carbonyl compounds that widely exist in biological systems are generally produced by free-radical-induced lipid peroxidation reaction.They have toxic effects on biological tissues.They can react with protein and amino acid,results in enzyme and cell membranes irreversible damage.Therefore,it’s important to detection of carbonyl compounds with high selectivity and sensitivity for the early clinical diagnosis.However,only targeted analysis of carbonyl compounds was generally performed in the previous methods,and non-targeted profiling of carbonyl compounds in the biological samples was rarely reported.MS is widely used for metabolites research due to its high sensitivity and specificity.However,certain compounds which absence of ionizable functional groups can’t be detected.In addition,mass spectrum signal can be influenced by matrix effects and instrument fluctuation.To resolve these problems,stable isotope internal standards can be used to overcome the matrix effects and ionization differences for the accurate quantification.However,few isotope standards are commercially available,which limit the application of masss spectrometry in some extent.In the early stage,stable isotope labeling(IL)strategy was applied in proteomics.Recently,it has been developed for targeted quantitative and non-targeted qualitative or quantitative profiling of metabolites with MS.IL method can introduce a light and heavy isotope tags to the two samples by chemical derivatization.Then the two labeled samples were mixed and subjected to LC-MS analysis.Quantitative analysis can be obtained by calculating the peak intensity ratios of the isotope labeled peak pairs in two comparative samples.However,in the traditional metabonomics analysis,high-resolution mass spectrometer(i.e.,Q-FOF or FTICR)is needed,which was expensive.While low-resolution instrument has the lower sensitivity,selectivity and accuracy.In this text,we used the IL strategy in combination with triple quadrupole mass spectrometer or LTQ-Orbitrap MS for analysis of carbonyl compounds in biological sample.The research contents were as follows:1.We developed a sensitive method for the detection of steroid hormones in follicular fluid by stable isotope labeling coupled with UPLC-ESI-MS/MS analysis.GP and ds-GP were used to label these steroid hormones.The d5-GP labeled standards were used as internal standards for quantification to minimize quantitation deviation in MS analysis due to the matrix and ion suppression effects.The ionization efficiencies of steroid hormones were greatly improved by 4-504 folds through the introduction of a permanent charged moiety of quaternary ammonium from GP.Using the developed method,we successfully quantified steroid hormones in human follicular fluid.We found that the contents of testosterone and androstenedione exhibited significant increase while the content of pregnenolone had significant decrease in follicular fluid of polycystic ovarian syndrome(PCOS)patients compared with healthy controls.2.We synthesized four derivatization reagents and evaluated them with their labeled products.with the four derivatization reagents labeling,the fragmentation behavior of the four labeled products was similar by each labeling reagent.The labeled products more likely to produce the same ions,which generated from the derivatization reagent moiety.We investigated the detection sensitivity of labeled products by using RPLC-MS both in the SIM and MRM mode.The detection sensitivity can be greatly improved through the introduction of a permanent charged moiety of quaternary ammonium from derivatization reagents.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.At last,we selected the 2-(2-hydrazinyl-2--oxoethyl)isoquinolin-2-ium bromide(HIQB)as derivatization reagent in the following experiments.3.Here,we developed an IL-LC-DPIS-MS method for comprehensive profiling of carbonyl compounds in human serum and an IL-LC-MRM-MS method for relative quantification of carbonyl compounds in myelogenous leukemia patients and healthy controls.For qualitative analysis,two equal volume of the pooled serum sample werelabeled with HIQB and d7-HIQB,respectively.Then the light and heavy labeled samples were mixed and analyzed by LC-DPIS-MS.The HIQB and d7-HIQB labeled carbonyl compounds could generate two characteristic product ions of 130.1/137.1 under collision-induced dissociation(CID),which contain an isotope tag and therefore were used for double precursor ion scans in mass spectrometry analysis.And they were recorded in two individual ion chromatograms.Peak-pair data were extracted from the two ion chromatograms(m/z 130.1 and 137.1)respectively,according to a mass shift of 7 Da,and only peak pairs with the same retention time and intensities were assigned to the potential candidates of carbonyl compounds.Using the IL-LC-DPIS-MS method,156 potential carbonyl compounds were found in human serum sample.The relative quantification of carbonyl compounds in serum samples between ML and healthy controls was performed by LC-MRM-MS in positive mode.The transitions of[M]+→130.1 and[M+7]+→137.1 for HIQB and d7-HIQB labeled carbonyl compounds,respectively,were used as MRM ion pairs.All the precursor ions([M]+ and[M+7]+)for the MRM quantification were obtained from the DPIS method in the serum samples.We further investigated the content changes of the 156 carbonyl compounds in serum samples of ML patients compared to healthy controls.44 carbonyl compounds were found to have significant difference between ML patients and healthy controls4.We developed an stable isotope labeling combining with high resolution mass spectrum(IL-Orbitrap-MS)method for comprehensive profiling of carbonyl compounds in mice feces.An equal volume of the pooled mice feces sample was labeled with HIQB and d7-HIQB,respectively.Then the light and heavy labeled samples were mixed and analyzed by IL-Orbitrap-MS.The same carbonyl compound labeled by the HIQB and d7-HIQB have the same retention time and intensities and have a mass shift of 7.043 Da.Based on this,we can obtained the potential candidates of carbonyl compounds.with this method,248 potential carbonyl compounds were found in mice feces sample,50 of which were further identified by commercial standards.