Current and Future Perspectives on the Structural Identification of Small Molecules in Biological Systems

Abstract

Although significant advances have been made in recent years, the structural elucidation of small molecules continues to remain a challenging issue for metabolite profiling. Many metabolomic studies feature unknown compounds; sometimes even in the list of features identified as "statistically significant" in the study. Such metabolic "dark matter" means that much of the potential information collected by metabolomics studies is lost. Accurate structure elucidation allows researchers to identify these compounds. This in turn, facilitates downstream metabolite pathway analysis, and a better understanding of the underlying biology of the system under investigation. This review covers a range of methods for the structural elucidation of individual compounds, including those based on gas and liquid chromatography hyphenated to mass spectrometry, single and multi-dimensional nuclear magnetic resonance spectroscopy, and high-resolution mass spectrometry and includes discussion of data standardization. Future perspectives in structure elucidation are also discussed; with a focus on the potential development of instruments and techniques, in both nuclear magnetic resonance spectroscopy and mass spectrometry that, may help solve some of the current issues that are hampering the complete identification of metabolite structure and function.

Keywords: Fourier transform-infrared spectroscopy; mass spectrometry; metabolite profiling; metabolomics; nuclear magnetic resonance spectroscopy; structure elucidation.

Figure 1

Nuclear magnetic resonance spectroscopy (NMR) of aqueous metabolites extracted from a sample of Daphnia magna. Complex overlapping peaks can be observed even in a shift range of just 0.5–5 ppm.

Figure 2

Section of a ¹H NMR of soil extract (black) overlaid with NMR spectra of lactate (red) and valine (blue).

Figure 3

2D COSY ¹H spectra of the Daphia sample from Figure 1 illustrating cross peaks.

Figure 4

Identification of molecular formula using uHRMS in combination with isotopic fine structure: (A) Full Scan MS⁺ spectra for a glutathione standard showing three calculated formula matches within 2 ppm mass error; (B) Expansion of m/z 309.07–309.13 showing M + 1 isotopologues and inability to determine parent formula; (C) Expansion of m/z 310.06–310.13 showing M + 2 isotopologues with calculated isotopic fine structure match of ³⁴S, ¹⁸O and ¹³C₂ isotopologues to C₁₀H₁₇N₃O₆S matching the glutathione standard, other formula fine structures do not match the same isotopologue profile.

Figure 5

Dereplication and identification strategy using Liquid Chromatography Mass Spectrometry: (A) Reverse Phase ESI⁺ Total Ion Chromatogram with overlaid Extracted Molecular Features of Canola Extract; (B) Extracted Ion Chromatogram of the molecular formula corresponding to the plant hormone methyl jasmonate with inlaid Find-by-Formula Spectrum showing observed and predicted isotopic abundance for the feature at 9.880–9.965 min; (C) Product Ion spectra of methyl jasmonate using collision voltage of 20.0 eV; (D) Spectral match of methyl jasmonate MS/MS spectra to the METLIN Database allowing Putative identification.

Figure 6

Comparison between the FTIR Response Spectra of E. coli and L. innocua at the surfactants concentrations of N-tetradecyltropinium bromide that resulted in 100% mortality. Note: a.u. stands for “arbitrary units”. Arrows correspond to the wavenumbers referred to peptidoglycan while circles to the wavenumbers referred to the major response peaks in each species. Blue, L. innocua; Red, E. coli. The synthetic stress of the metabolomic stress response are defined by the following FTIR regions: fatty acids (W1) from 3000 to 2800 cm⁻¹, amides (W2) from 1800 to 1500 cm⁻¹, mixed region (W3) from 1500 to 1200 cm⁻¹, carbohydrates (W4) from 1200 to 900 cm⁻¹ and typing region (W5) from 900 to 700 cm⁻¹. Adapted from Corte et al. [125].