Metabolic profiling has received increasing recognition as an essential complement to genomics and proteomics for probing biological systems and for clinical applications. 1H nuclear magnetic resonance (NMR) spectroscopy is widely used in the field. Currently, 1H NMR spectroscopy is challenged by spectral complexity, overlap and relatively poor sensitivity. At Purdue, focused on circumventing such bottlenecks, new NMR methods are being developed. Two such methods combine 15N or 13C isotope tagging of carboxyl or amino group containing metabolites and detection by 1H-15N HSQC or 1H-13C HSQC NMR experiments, respectively [Figure 1]. Here 15N or 13C isotope tags are reacted with carboxyl or amino group containing metabolites in complex biological mixtures using simple chemical derivatization reactions.
Figure 1: 15N and 13C isotope tagging of carboxyl and amino group containing metabolites in biofluids and their detection by 2D NMR.
This new approach achieves detection of nearly 10-fold increase in the number of quantifiable metabolites with significantly improved resolution and sensitivity, which is unprecedented for NMR spectroscopy-based metabolites detection, and paves new ways for metabolic profiling applications. Advantages of this approach include quantitative accuracy, improved resolution that reduces spectral overlap, and a concomitant improvement in unknown peak identification. As shown below, for example, the 2D NMR spectra of urine and serum obtained after tagging carboxyl group containing metabolites with 15N isotope are very rich, with a large number of peaks (close to 200) that correspond to the individual acid groups that have been converted to 15N labeled amides [Figure 2]. The spectra are very reproducible, and the signals are linear in response to changes in metabolite concentration. Limits of detection for this approach are in the low micro molar range, which is lower than for 1D NMR experiments due to the high degree of overlap in the 1D NMR spectra.
 
Figure 2: 2D 1H-15N HSQC spectrum of human urine (left) and serum (right) obtained after tagging carboxyl group containing metabolites with 15N-ethanolamine. Nearly 200 metabolite signals are seen in each spectrum and a number of these have been identified by comparing the chemical shifts with PIE-NMR Database.
Carboxyl and amino group containing compounds represent a large and important class of metabolites or exogenous compounds, and they appear in essentially all important metabolic pathways. A number of these compounds are known biomarkers or potential biomarkers for various diseases. Simultaneous detection of a class of metabolites of this large number, we believe, will be of high utility towards the advancement of metabolic profiling.
References
Shanaiah N, Desilva A, Nagana Gowda GA, Raftery MA, Hainline BE, Raftery D. Metabolite class selection of amino acids in body fluids using chemical derivatization and their enhanced 13C NMR. Proc. Natl. Acad. Sci (USA), 104(28), 11540-11544 (2007).
Ye T, Mo H, Shanaiah N, Nagana Gowda GA, Zhang S, Raftery D. Chemoselective 15N Tag for Sensitive and High-Resolution Nuclear Magnetic Resonance Profiling of the Carboxyl-Containing Metabolome, Anal. Chem., 81(12), 4882-4888 (2009).
Ye T, Zhang S, Mo H, Tayyari F, Nagana Gowda GA, Raftery D. 13C-Formylation for Improved NMR Profiling of Amino Metabolites in Biofluids, Anal. Chem., 82(6), 2303-2309 (2010).
Nagana Gowda GA, Tayyari F, Ye T, Suryani Y, Wei S, Shanaiah N, Raftery D. Quantitative Analysis of Blood Plasma Metabolites Using Isotope Enhanced NMR Methods, Anal. Chem., 82(21), 8983-8990(2010).
|
