InterSpin: Integrated Supportive Webtools for Low- and High-Field NMR Analyses Toward Molecular Complexity

Abstract

InterSpin (http://dmar.riken.jp/interspin/) comprises integrated, supportive, and freely accessible preprocessing webtools and a database to advance signal assignment in low- and high-field NMR analyses of molecular complexities ranging from small molecules to macromolecules for food, material, and environmental applications. To support handling of the broad spectra obtained from solid-state NMR or low-field benchtop NMR, we have developed and evaluated two preprocessing tools: sensitivity improvement with spectral integration, which enhances the signal-to-noise ratio by spectral integration, and peaks separation, which separates overlapping peaks by several algorithms, such as non-negative sparse coding. In addition, the InterSpin Laboratory Information Management System (SpinLIMS) database stores numerous standard spectra ranging from small molecules to macromolecules in solid and solution states (dissolved in polar/nonpolar solvents), and can be searched under various conditions using the following molecular assignment tools. SpinMacro supports easy assignment of macromolecules in natural mixtures via solid-state ¹³C peaks and dimethyl sulfoxide-dissolved ¹H-¹³C correlation peaks. InterAnalysis improves the accuracy of molecular assignment by integrated analysis of ¹H-¹³C correlation peaks and ¹H-J correlation peaks of small molecules dissolved in D₂O or deuterated methanol, which supports easy narrowing down of metabolite candidates. Finally, by enabling database interoperability, SpinLIMS's client software will ultimately support scientific discovery by facilitating sharing and reusing of NMR data.

Figure 1

Overview of InterSpin. InterSpin is a freely accessible integrated supportive webtool for advanced performance of NMR signal assignment in low- and high-field NMR analyses of small- to macromolecular mixtures. InterSpin comprises the following three elements. (1) Spectrum-preprocessing tools. In the case of a broad spectrum obtained from low-field benchtop ¹H-NMR or solid-state ¹³C CP-MAS, sensitivity improvement with spectral integration (SENSI) helps to overcome the problem of low signal-to-noise ratio by increasing resolution through the integration of multiple spectra, whereas PKSP supports effective peak separation by a multivariate spectral decomposition method. (2) Molecular assignment tools. SpinMacro supports simplifying the macromolecular assignment of a solid CP-MAS spectrum or a DMSO-solubilized ¹H–¹³C HSQC spectrum. SpinAssign searches the SpinLIMS database for a compound corresponding to the HSQC NMR peaks. SpinCouple can assign ¹H–J 2D-J_res NMR peaks. InterAnalysis is a Venn-diagram-type highly accurate annotation tool that helps to narrow down candidate molecules using correlation peaks from both the HSQC spectrum and the 2D-J_res spectrum. In the bottom right of the figure, blue, yellow, and red circles represent a set of search results; the green star represents the narrowed-down set. (3) InterSpin Laboratory Information Management System (SpinLIMS) database. The database includes reference solid-state CP-MAS spectra and solution-state HSQC spectra (DMSO) for macromolecules, and reference solution-state HSQC and 2D-J_res spectra (D₂O and MeOD) for small molecules.

Figure 2

Comparison of the analysis speed of each algorithm in peaks separation (PKSP). (a) Three average analysis times for 25, 50, 100, and 198 components (i.e., compounds to be separated by each algorithm of PKSP). (b) Three average analysis speeds for 25, 50, 100, and 198 components.

Figure 3

Molecular assignment of a mixture using peaks separated by PKSP (NNSC). (a) Original and separated spectra of No. 33 Thunnus sample measured by benchtop 60 MHz NMR. Original and separated component spectra of (b) histidine, (c) creatine, and (d) lactate.

Figure 4

How to assign macromolecules in a mixture using “SpinMacro”. The flow of data through SpinMacro is shown. The user queries of CP-MAS peaks or HSQC peaks are entered as PHP. The SpinLIMS database is then searched for candidate molecules within the set range of ¹³C chemical shifts for CP-MAS, or ¹H and ¹³C chemical shifts for HSQC.

Figure 5

CV of peaks picked by SENSI from E. gracilis CP-MAS spectrum. (a) SENSI results. Red circles are the picked peaks. The enlarged view (top left) shows the raw spectrum of paramylon from the data used for SENSI of a sugar region. (b) CV of peaks picked by SENSI. Blue circles indicate lipid signals, black circles indicate peptide signals, and red circles indicate paramylon signals.

Figure 6

Result of InterAnalysis for ¹H–¹³C HSQC and ¹H–J 2D-J_res peaks from Acanthogobius flavimanus body muscle extract in MeOD. The summary shows the number of query peaks, the number of assigned molecules, and the narrowed-down set of molecules. The table shows some of the molecular assignment results for each query peak.