I'm going to conduct an experiment for following the fate of labeled [1,2-13C2] glucose in TCA cycle using derivatzaion method by LCMS. Since the lable Carbons internaly pass on to the TCA metabolite, do I need to use internal standards (standard isotope) for sample preparation? If no, In which way I can analysis the TCA intermediates? Thank you!
There are several questions that need to be clarified first...
Is this a targeted analysis, I mean do you know which compounds you need to follow in the TCA cycle and which compounds will get the labeled carbons (which carbons and how many of them, etc..) ?... If so targeted metabolomic study is intended and the intermediates you investigated must be quantified by using matrix-matched calibration spiked with isotope-labeled versions of the intermediates...This is the absolute quantification strategy you may perform on the TQ-MS system...If absolute quantification is not needed similar ionization efficiency can be obtained from labeled and unlabeled forms of the target compound on ESI-MS and thus you may calibrate your instrument by using only the intact version of the TCA intermediates you follow...
Historically, radioisotopic methods were used where each intermediate was isolated and quantified. Stable isotopic methods, using MS and MS/MS have displaced this (see attached MetaSIRMS brochure). Isotopic vector angle analysis is the best method to do this for which there are papers by myself and Price at BYU that describe the method. This will only work for intermediates that are ionizable. There are similar C-NMR methods by a researcher in France (sorry can't remember the name) which can be used for non-ionizable compound analysis. Isotopic ratio methods work best because compound recovery can be assumed to be the same for both isotopic versions. However, you always have to be aware of enzymatic isotopic preferences, even for 13C, in metabolism. Therefore, its best to use low ratios of stable isotopes, rather than pure isotopic labels, otherwise growth rates are low, or the organism can fail to grow, even bacteria.
Method Isotopic Vector Angle Analysis in Mass Spectrometry v2
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