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An Integrated Analytical and Statistical Two-Dimensional Spectroscopy Strategy for Metabolite Identification: Application to Dietary Biomarkers Joram M. Posma, Isabel Garcia-Perez, James Christopher Heaton, Paula Burdisso, John C. Mathers, John Draper, Matthew R. Lewis, John C Lindon, Gary S. Frost, Elaine Holmes, and Jeremy Kirk Nicholson Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03324 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on March 1, 2017

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Analytical Chemistry

JOURNAL Analytical Chemistry (ac-2016-03324s.R2) TITLE An Integrated Analytical and Statistical Two-Dimensional Spectroscopy Strategy for Metabolite Identification: Application to Dietary Biomarkers AUTHOR NAMES Joram M. Posma,†,⊥ Isabel Garcia-Perez,†,‡,⊥ James C. Heaton,†,∆ Paula Burdisso,†,# John C. Mathers,§ John Draper,ǁ Matt Lewis,†,∩ John C. Lindon,† Gary Frost,‡ Elaine Holmes,†,∩,* and Jeremy K. Nicholson†,∩,* AUTHOR ADDRESS † Section of Biomolecular Medicine, Division of Computational and Systems Medicine, Department of Surgery and Cancer, Faculty of Medicine, South Kensington Campus, Imperial College London, London, SW7 2AZ, United Kingdom; ‡ Nutrition and Dietetic Research Group, Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Faculty of Medicine, Hammersmith Campus, Imperial College London, London, W12 ONN, United Kingdom; § Human Nutrition Research Centre, Institute of Cellular Medicine, Newcastle University, United Kingdom; ǁ Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, United Kingdom; ∩ MRC-NIHR National Phenome Centre, Department of Surgery and Cancer, Faculty of Medicine, Hammersmith Campus, Imperial College London, London, W12 0NN, United Kingdom KEYWORDS Analytical Framework, Bayesian Analysis, Controlled Clinical Trial, Dietary Biomarkers, Food Challenge, Gaussian Mixture Model, Markov-Chain Monte-Carlo, Statistical Spectroscopy

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ABSTRACT A major purpose of exploratory metabolic profiling is for the identification of molecular

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species that are statistically associated with specific biological or medical outcomes;

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unfortunately the structure elucidation process of unknowns is often a major bottleneck in

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this process. We present here new holistic strategies that combine different statistical

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spectroscopic and analytical techniques to improve and simplify the process of metabolite

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identification. We exemplify these strategies using study data collected as part of a dietary

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intervention to improve health and which elicits a relatively subtle suite of changes from

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complex molecular profiles. We identify three new dietary biomarkers related to the

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consumption of peas (N-methyl nicotinic acid), apples (rhamnitol) and onions (N-acetyl-S-

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(1Z)-propenyl-cysteine-sulfoxide) that can be used to enhance dietary assessment and assess

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adherence to diet. As part of the strategy, we introduce a new probabilistic statistical

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spectroscopy tool, RED-STORM (Resolution EnhanceD SubseT Optimization by Reference

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Matching), that uses 2D J-resolved 1H-NMR spectra for enhanced information recovery using

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the Bayesian paradigm to extract a subset of spectra with similar spectral signatures to a

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reference. RED-STORM provided new information for subsequent experiments (e.g. 2D-

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NMR spectroscopy, Solid-Phase Extraction, Liquid Chromatography prefaced Mass

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Spectrometry) used to ultimately identify an unknown compound. In summary, we illustrate

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the benefit of acquiring J-resolved experiments alongside conventional 1D 1H-NMR as part

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of routine metabolic profiling in large datasets and show that application of complementary

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statistical and analytical techniques for the identification of unknown metabolites can be used

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to save valuable time and resource.

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INTRODUCTION

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Dietary interventions (DIs) are a cornerstone in the management of reducing the risk of non-

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communicable diseases1-3 and promoting healthy aging4. However, understanding the

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response to dietary change is compromised by poor compliance to dietary recommendations

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and the inherently inaccurate self-reporting dietary recording tools available, with prevalence

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of misreporting estimated at 30–88%5,6, lowering the value of such studies and data

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It has been demonstrated that dietary biomarkers can reflect consumption of specific

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foods and enhance dietary intake assessment at individual and population levels7-17. Dietary

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biomarkers are based on the concept that food intake is highly correlated with excretion

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levels of food-related compounds over a given period of time. These ‘biomarkers’ can be

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compounds that are excreted unchanged10,17 or that have undergone metabolic conversion for

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example by gut bacteria8,11,13,14. Metabolic profiling of biofluids using spectroscopic

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technologies18 can detect thousands of compounds simultaneously, generating a profile that

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can be related to specific states of health or disease. Some of the compounds in these spectral

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profiles are potential biomarkers of dietary intake. However, finding associations between

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self-reported dietary intakes and excreted metabolites19,20 in order to discover potential food

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biomarkers is plagued by inaccuracies of self-reporting. Thus, confirmation of a candidate

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biomarker is best achieved by using a controlled food challenge with subsequent validation in

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a larger population or study cohort.17 The complementarity of the main workhorses of metabolic profiling, one-dimensional

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Nuclear

Magnetic

Resonance

(1H-NMR)

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proton

spectroscopy

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chromatograpic-mass spectrometry (MS) techniques, has been extensively demonstrated in

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the last decade21-23. However, chemical characterization of molecular species associated with

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an outcome still is a limiting factor of exploratory metabolic profiling. NMR spectroscopy

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provides an atom-centered spectroscopic tool for structure elucidation that can be enhanced 3 ACS Paragon Plus Environment

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by statistical spectroscopic methods24,25 or by physical hyphenation with chromatographic

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methods such as Solid-Phase Extraction (SPE)26, Liquid Chromatography (LC)21 or by LC-

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NMR-MS21,27 to achieve a better chemical characterization of endogenous and exogenous

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metabolites.

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Metabolite identification in 1H-NMR spectroscopy is aided by the intrinsic correlation

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of peaks from the same metabolite. Statistical TOtal Correlation SpectroscopY (STOCSY)28

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makes use of this property by calculating the correlation between one spectral variable

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(driver) and all other variables to uncover structural associations. In cases where there are

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sufficient spectra, identification of 1H-NMR peaks using statistical methods is an efficient

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strategy that utilizes existing spectral data without requiring additional spectroscopic

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experiments a priori, which has obvious advantages in usage of volume-limited samples and

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is cost-efficient.23 Since the STOCSY method was published, many derivations have aimed to

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improve specific properties such as differentiation between structural and pathway

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correlations by clustering, subset selection or stoichiometric relationships.24

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Statistical correlation can be undermined by overlapped signals unrelated to the

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metabolite of interest in a 1D-NMR spectrum and 2D-NMR experiments are still required for

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unambiguous structure elucidation29. In addition, the structural information obtained using

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statistical algorithms is dependent on criteria such as correlation thresholds28,30 or correlation-

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distance cut-offs31. SubseT Optimization by Reference Matching (STORM)32 is a derivation

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of STOCSY that aims to separate out confounding spectra that do not match a supplied

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reference spectrum of a potential biomarker signal, thereby showing clearer spectral

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correlations between variables for both low and high intensity signals. The reference

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spectrum is a single spectrum that contains the signal of interest. The peak segment is

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correlated with the same region of all samples and a high correlation indicates the samples

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contain the same signal and are likely ‘informative’, whereas samples with a low correlation 4 ACS Paragon Plus Environment

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do not have this signal and are uninformative. Subsets of spectra and variable correlations are

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found by carefully correcting for multiple testing in both phases and using statistical

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shrinkage. Here, we describe a holistic strategy for the identification of unknown metabolites

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that combines the strengths of statistical spectroscopy, NMR, multiple separation techniques

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and MS, and apply the strategy to identify three novel dietary biomarkers. In addition, we

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demonstrate an extension of STORM to 2D-NMR spectra to uncover the identity of unknown

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metabolites.

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EXPERIMENTAL SECTION

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Food challenges (FCs) – To discover urinary biomarkers of peas, apples and onions three

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FCs were designed. A total of nine healthy participants (4 women, 5 men; non-smokers; age:

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22–32y; BMI: 21.2–25.3 kg/m2) were recruited and assigned to one of the three FCs.

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Participants were provided with the assigned foods as part of a standardized dinner (incl.

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125g of chicken breast as a protein source). Incremental amounts of the designated food were

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consumed over three consecutive days: 60/120/180g for (boiled) peas, 40/80/160g for (raw)

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apples and 20/40/60g for (fried) onions. For 24hr preceding the FC, and throughout the FC,

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participants were asked to consume their habitual diet and avoid consumption of

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coffee/tea/cocoa and any additional amounts of assigned foods. Cumulative urine samples

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were collected into sterilized single-use urine containers (International Scientific Supplies

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Ltd., Bradford, U.K.) from dinner up to and including the first morning void. Urine samples

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were stored at −80°C until analysis.

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Controlled clinical trial (CCT) – 19 healthy participants (9 women, 10 men; non-smokers;

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age: 25–60y; BMI: 21.1-33.3 kg/m2) attended the NIHR/Wellcome Trust Imperial Clinical

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Research Facility for four 3-day inpatient periods, separated by a period of >4 days, with

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food and drink intake tightly controlled (alcohol/coffee/tea/cocoa were not provided). In 5 ACS Paragon Plus Environment


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random order, participants followed all four DIs representing 100% (Diet 1), 75% (Diet 2),

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50% (Diet 3) and 25% (Diet 4) of WHO healthy eating guidelines1 with respect to

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carbohydrates, fats, fiber, fruits, salt sugar and vegetables. Full details of the clinical trial

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design have been described previously33. Foods consumed relevant to the present study are

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tabulated in Supporting Information.

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Each participant collected cumulative urine samples (CS) on each day of each DI

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from after breakfast to before lunch (CS1), after lunch to before dinner (CS2) and after dinner

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to before breakfast the following day (CS3). 24hr urine samples were obtained by pooling the

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cumulative samples. Aliquots of urine were transferred into Eppendorf tubes and stored at

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−80°C until analysis. All participants provided informed, written consent prior to the CCT

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(Registration No: ISRCTN-43087333), which was approved by the London Brent Research

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Ethics Committee (13/LO/0078). All studies were carried out in accordance with the

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Declaration of Helsinki.

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1

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for 5min. All available samples (nFC=27, nCCT=906, for missing CCT data see Supporting

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Information) were prepared for 1H-NMR spectroscopy following the protocol described in34,

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mixing 540µL of supernatant with 60µL of pH7.4 phosphate buffer containing trimethylsilyl-

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[2,2,3,3,-2H4]-propionate as an internal reference standard (‘NMR-buffer’). Water-suppressed

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1

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Biospin,

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(RD−gz,1−90°−t−90°−tm−gz,2−90°−ACQ) with saturation of the water resonance7. RD is the

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relaxation delay, t is a short delay (4µs), 90° represents a radio frequency (RF) pulse that tips

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the magnetization by 90°, tm is the mixing time (10ms), gz,1 and gz,2 are magnetic field z-

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gradients both applied for 1ms, and ACQ is the data acquisition period of 2.73s. 1H-NMR

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spectra were acquired using 4 dummy scans and 32 scans, 64K time domain points and with a

H-NMR analysis – Aliquots of 600µL of urine samples were centrifuged at 16,000xg at 4°C

H-NMR spectroscopy was performed at 300K on a Bruker 600MHz spectrometer (Bruker Karlsruhe,

Germany)

using

a

standard


1D

pulse

sequence

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spectral window of 20ppm. Prior to Fourier transformation, free induction decays were

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multiplied by an exponential function corresponding to a line broadening of 0.3Hz. 1H-NMR

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spectra were normalized to the total urine volume to correct for differences in dilution.

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H–1H 2D J-resolved experiments7 were acquired using a pulse sequence to detect the

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J-couplings in the second dimension, with suppression of the water resonance (RD–90°–t1–

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180°–t1–ACQ), where t1 is an incremented time period, RD is 2s and 180° represents a 180°

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RF pulse, ACQ is 0.41s. J-resolved spectra were acquired using 16 dummy scans and 2 scans,

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8K points with spectral window of 16.7ppm for f2 and 40 increments with spectral window

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of 78Hz for f1. Continuous wave irradiation was applied at the water resonance frequency

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using a 25Hz RF during the RD. A sine-bell apodization function was applied to f2 and a

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squared sine-bell to f1 of the J-resolved data, followed by Fourier transformation, tilting by

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45° and symmetrization along f1 before data analysis.

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A suite of 2D-NMR experiments including 1H–1H TOtal Correlation SpectroscopY 1

H–1H COrrelation SpectroscopY (COSY),

1

H–13C Hetero-nuclear Single

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(TOCSY),

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Quantum Coherence (HSQC) and

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(HMBC) spectroscopy were used for identification purposes25,29.

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SPE-NMR – Apple extracts were homogenized using a Kenwood KMix Blender for 5min.

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The puree obtained was filtered using a stainless steel filter and centrifuged for 10min at

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16,000xg. 2mL of each sample (urine/apple) were lyophilized overnight. Freeze-dried (FD)

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urine/apple samples were dissolved in 1mL of 50mM sodium phosphate pH8.5 and briefly

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sonicated prior to being loaded onto a 100 mg/ml Bond Elut™ phenylboronic acid (PBA)

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SPE-cartridge (Agilent Technologies, Stockport, U.K.). The SPE-cartridge was conditioned

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with 1mL of 70:30v/v H2O:ACN, 0.1M HCl followed by 1mL of 50mM sodium phosphate

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pH10. After conditioning, the sample was loaded onto the SPE-cartridge. The loaded sample

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was washed with 2mL 50:50v/v ACN:sodium phosphate (10mM) pH8.5. The sample was

1

H–13C Hetero-nuclear Multiple-Bond Correlation



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eluted using 1mL of acidified solutions (0.1M HCl) of 100% H2O, 90:10v/v H2O:ACN and

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70:30v/v H2O:ACN and subsequently lyophilized until dry. The dried eluent fractions were

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reconstituted in 540µL H2O and 60µL of NMR-buffer, vortexed and centrifuged prior to

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NMR analysis.

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LC-NMR-MS27 – 5mL of urine collected overnight after consumption of onion was

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lyophilized overnight, reconstituted in 500µL of the original urine sample and vortexed,

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sonicated and centrifuged (20min at 16,000xg). The supernatant was repeatedly injected

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(7×2µL) onto a reversed-phase HPLC column (Waters Atlantis-T3, 3µm, 4.6×150mm at

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30°C) in a Waters Acquity UPLC comprising a binary solvent manager and photodiode array

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detector with a Waters CTC auto-sampler with 100µL sampling needle, and eluted at

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0.8mL/min using the following gradient: 0.0–60.0min (99.9:0.1% H2O/formic acid), 60.01–

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65.0min (99.9:0.1% methanol/formic acid), 65.01–127.5min (99.9:0.1% H2O/formic acid).

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The chromatographic separation of the sample was fractionated using a Waters Fraction

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Collector III. A total of 120 fractions were collected, one every 29s (starting at t=5min,

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finishing at t=63min), and dried under a stream of nitrogen. Each fraction was re-dissolved in

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540µL of H2O and 60µL of NMR-buffer and analyzed by 1H-NMR. A volume of 50µL of the

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fraction containing the unknown metabolite was analyzed by reversed-phase LC-MS, Waters

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Acquity Ultra Performance LC system coupled to Xevo G2 Q-TOF mass spectrometer

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(Waters, Milford, MA, U.S.A.), following an established metabolic profiling method35. The

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optimized capillary voltage, cone voltage and collision energy were 3kV/20V/4V for ESI+

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and 1.5kV/30V/6V for ESI-, using a source temperature of 120°C and desolvation

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temperature of 600°C. Desolvation was 1000L/h for both and cone gas flows 50L/h (ESI+)

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and 150L/h (ESI-). Leucine enkephalin was used as the reference lock mass at 556.2771

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([M+H]+) and 554.2615 ([M-H]-).


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RED-STORM algorithm – Here, STORM was extended in a probabilistic framework and

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modified for applicability to 2D data to provide a clearer signature of structural correlations

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in the data and the extended algorithm was named Resolution EnhanceD SubseT

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Optimization by Reference Matching (RED-STORM).

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High correlations in the data are of interest between samples and a reference spectrum

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of a signal of interest (for subset optimization) and between a driver and all other variables

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(for assessing variable importance). However, high correlations are not normally distributed

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because of the upper bound on the correlation ([-1,1]), this results in their distributions being

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negatively skewed. Therefore, the correlation () is transformed to a Fisher z-score, which

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results in approximately normally distributed data that can be analyzed using parametric

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methods:

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1.

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Next, the distribution of all z-scores is modelled using a Gaussian Mixture Model (GMM). A

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GMM is a weighted sum of k Gaussian clusters and is defined as

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2.

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Where has data points, are the means, the variances, the mixture weights for

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= ln

|, , = ∑ , ,

each of Gaussians ( ∑ = 1 ) and , a normalized Gaussian distribution with

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specified and . In order to obtain clusters of variables from the Fisher z-transformed

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correlations a parameter-free GMM is used36. The model automatically learns the optimal

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number of clusters from the data. For completeness a description of the method in brief

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follows, for mathematical proofs see36.

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Each cluster mean ( ), and precision ( ) from equation 2 are given Gaussian

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( |", # ∼ , ", # ) and Gamma ( |%, & ∼ Γ%, & ) priors, respectively. Here, " and # are hyperparameters for the means of all clusters. Similarly, % (shape) and & 9

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(scale) are hyper-parameters for the cluster precisions. The hyper-parameters themselves are

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initialized from the observation mean (( ) and variance (( ) as vague priors ( " ∼

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, ( , ( ; # ∼ Γ1, ( ; % ∼ Γ1,1 ; & ∼ Γ1, ( ). The posterior

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distributions of the cluster means are obtained from

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3.

229

(,- .-/ 01

,

2- .-/ 1 2- .-/ 1

3

̃ = ∑87 456, 7

with

and

* =

∑87 456 ,

227

228

|̃ , * , , ", # ∼ , +

where ̃ is the sum of observations from cluster 9 , * the number of observations with highest probability for cluster 9, :7 the cluster with highest probability for observation ; and

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456, a Kronecker delta product. Similarly, the posterior distributions of the cluster precisions

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are obtained from

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4.

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The posteriors for " ( "|, # ∼ , +

234

1,

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the priors, whereas posterior for % takes a different form and makes use of the fact that

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>? @

|* , %, & ∼ Γ 2 - 3 A with B = ∑87 456 , 7 − -

DE .EF/ 1 ∑G -HI D-

.E/ ∑G -HID- 0

/

.EF/ 1

A) and & (&| , % ∼ Γ +% + 1,

, .F/ 13 ), # ( #|, " ∼ Γ

F/ .EF/ > ∑G -HI .-

3) are obtained similarly to

J%| , & is log-concave36: %|

>

, & ∝ Γ

>

LM >

GNFO /

N

>.-F/ ?

∏

An Integrated Analytical and Statistical Two-Dimensional

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