Stable-Isotope Trapping and High-Throughput Screenings of Reactive Metabolites Using the Isotope MS Signature

Abstract

A highly efficient method has been developed to detect and identify reactive metabolites, using stable-isotope trapping combined with ESI-MS/MS neutral loss scanning. A mixture of glutathione (GSH, gamma-glutamylcysteinylglycine) and the stable-isotope labeled compound (GSX, gamma-glutamylcysteinylglycine-(13)C(2)-(15)N) was used at an equal molar ratio to trap reactive metabolites generated in microsomal incubations. Samples resulting from incubations were cleaned and concentrated by SPE, followed by LC-MS/MS analyses using constant neutral loss scanning for 129 Da (the gamma-glutamyl moiety) to detect formed GSH conjugates. Unambiguous identification of glutathione adducts was greatly facilitated by the presence of a unique MS signature of a prominent isotopic doublet that differs in mass by 3 Da. Further structural characterization of conjugates was achieved with high confidence by subsequently acquiring MS/MS spectra that were featured by neutral losses of 75 and 129 Da for GSH adducts and 78 and 129 Da for isotopic GSX adducts. The reliability of this method was vigorously validated using a number of compounds known to form reactive metabolites. Superior sensitivity was demonstrated by the capability of the current approach to identify reactive metabolites at low abundance. Because of the unique isotopic MS signature, ultrafast analyses of reactive metabolites were accomplished by direct injection of cleaned samples into mass spectrometers for neutral loss scanning. More importantly, this study has demonstrated the feasibility of the current method for completely automated detection of reactive metabolites via computer-assisted pattern recognition.

Simultaneous Screening of Glutathione and Cyanide Adducts Using Precursor Ion and Neutral Loss Scans-Dependent Product Ion Spectral Acquisition and Data Mining Tools

“…A number of derivatives of GSH such as GSH ethyl ester, dansyl-GSH, brominated GSH, or quaternary ammonium GSH have also been utilized to facilitate MS detection or quantitation [13][14][15][16]. Introduction of stable isotope-labeled GSH analogues such as 13 C 2 15 N-GSH into an incubation medium provides GSHtrapped reactive metabolites with unique isotope patterns that can be used either in isotope pattern-dependent scan or post-acquisition data mining [17][18][19][20]. Hard reactive intermediates cannot be efficiently trapped by GSH or its derivatives.…”

“…Detection of GSH and CN adducts by triple quadrupole or triple quadrupole-linear ion trap (Qtrap) mass spectrometry is based on their unique fragmentation patterns. For example, neutral loss (NL) of 129 Da in the positive ion mode that corresponds to loss of pyroglutamic acid is the most commonly used scanning function for detection of GSH adducts by triple quadrupole instruments [18,24]. Alternatively, GSH adducts produce characteristic product ion at m/z 272 in negative mode, which can be used in precursor ion (PI) scan for detection [25].…”

Exposure Marker Discovery of Phthalates Using Mass Spectrometry

“…Various methods have been developed to perform objective searching/ ltering of accurate-mass-based LC-HRMS data to facilitate metabolite detection, such as isotope pattern lters (IPF), 42) mass defect ltering (MDF), 35,43) XCMS Online, 44) background subtraction, 45,46) and knowledgebased metabolic predictions. 47) A combination of di erent analytical technologies and data mining tools is needed to e ciently and thoroughly identify metabolites for discovering the biomarkers of exposure, toxic e ects, or diseases.…”

“…To achieve that, IPF is a stable isotope-based metabolomics approach using compound labeled with 2 H, 13 C, 15 N, or 18 O that can be used to facilitate identi cation of the metabolites. 42,48,49) e stable isotope ratio (1 : 1) from spiked native and isotope-labeled compounds were used for metabolite signal detection.…”

A statistical procedure to selectively detect metabolite signals in LC-MS data based on using variable isotope ratios

“…Results obtained here suggest that the statistical procedure could be used to confidently facilitate the detection of probable metabolites from a compoundderived precursor presented in a complex LC-MS dataset. One strategy to mine MS data for metabolite signals uses the isotope cluster that is formed after treating test subjects with an equal mixture of native and isotopelabeled compounds [1][2][3]. In mineral metabolism research, the stable isotope ratio (1:1) from spiked native and isotope-labeled compounds has been used for metabolite signal detection.…”