Use of optimized 1D TOCSY NMR for improved quantitation and metabolomic analysis of biofluids

Abstract

One dimensional selective TOCSY experiments have been shown to be advantageous in providing improved data inputs for principle component analysis (PCA) (Sandusky and Raftery 2005a, b). Better subpopulation cluster resolution in the observed scores plots results from the ability to isolate metabolite signals of interest via the TOCSY based filtering approach. This report reexamines the quantitative aspects of this approach, first by optimizing the 1D TOCSY experiment as it relates to the measurement of biofluid constituent concentrations, and second by comparing the integration of 1D TOCSY read peaks to the bucket integration of 1D proton NMR spectra in terms of precision and accuracy. This comparison indicates that, because of the extensive peak overlap that occurs in the 1D proton NMR spectra of biofluid samples, bucket integrals are often far less accurate as measures of individual constituent concentrations than 1D TOCSY read peaks. Even spectral fitting approaches have proven difficult in the analysis of significantly overlapped spectral regions. Measurements of endogenous taurine made over a sample population of human urine demonstrates that, due to background signals from other constituents, bucket integrals of 1D proton spectra routinely overestimate the taurine concentrations and distort its variation over the sample population. As a result, PCA calculations performed using data matrices incorporating 1D TOCSY determined taurine concentrations produce better scores plot subpopulation cluster resolution.

Can NMR solve some significant challenges in metabolomics?

“…For 1D, selective TOCSY is one such approach that has been shown to be useful for unraveling the complexity of NMR spectra. Selective TOCSY opens avenues for both unknown metabolite identification as well as reliable metabolite quantitation [40–42]. The ability of selective TOCSY to connect peaks that are part of the same coupled network of spins arising from a single metabolite results in multiple peaks that are free of interferences and thus aids significantly in positive identification.…”

“…TOCSY can also be used to improve the quantitation of highly overlapped metabolites in complex mixtures, as we demonstrate in Fig. 9d and e, for the case of taurine in urine [40]. And an additional advantage of selective TOCSY is that it enables the identification of low concentration metabolites that may be important for distinguishing sample classes even when they occur in the presence of more dominant, high intensity signals that do not provide such distinguishing capabilities.…”

NMR-based metabolomics: From sample preparation to applications in nutrition research

“…For example, the application of 1D selective TOCSY has been shown to help with the issue of overlapping peaks and thereby increase resolution [16]. More recently, a fast NMR method has been developed that involves the acquisition of three 2-D NMR spectra: these then provide complementary information to aid metabolite assignment [17].…”

Isotope Enhanced Approaches in Metabolomics

“…The 1D selective TOCSY (total correlation spectroscopy) methodology was explored for the analysis of metabolites in biofluids and was shown to detect metabolites quantitatively, even at concentrations much lower than those of the major components [38-40]. In a 1D selective TOCSY experiment, protons signals of interest are selectively excited by a shaped soft pulse; the magnetization is then transferred to all sequentially coupled spin system through J -couplings.…”