Practical Aspects of NMR-Based Metabolomics.

While NMR-based metabolomics is only about 20 years old, NMR has been a key part of metabolic and metabolism studies for >40 years. Historically, metabolic researchers used NMR because of its high level of reproducibility, superb instrument stability, facile sample preparation protocols, inherently quantitative character, non-destructive nature, and amenability to automation. In this chapter, we provide a short history of NMR-based metabolomics. We then provide a detailed description of some of the practical aspects of performing NMR-based metabolomics studies including sample preparation, pulse sequence selection, and spectral acquisition and processing. The two different approaches to metabolomics data analysis, targeted vs. untargeted, are briefly outlined. We also describe several software packages to help users process NMR spectra obtained via these two different approaches. We then give several examples of useful or interesting applications of NMR-based metabolomics, ranging from applications to drug toxicology, to identifying inborn errors of metabolism to analyzing the contents of biofluids from dairy cattle. Throughout this chapter, we will highlight the strengths and limitations of NMR-based metabolomics. Additionally, we will conclude with descriptions of recent advances in NMR hardware, methodology, and software and speculate about where NMR-based metabolomics is going in the next 5-10 years.

MagMet: A fully automated web server for targeted nuclear magnetic resonance metabolomics of plasma and serum.

Nuclear magnetic resonance (NMR) spectral analysis of biofluids can be a time-consuming process, requiring the expertise of a trained operator. With NMR becoming increasingly popular in the field of metabolomics, there is a growing need to change this paradigm and to automate the process. Here we introduce MagMet, an online web server, that automates the processing and quantification of 1D 1 H NMR spectra from biofluids-specifically, human serum/plasma metabolites, including those associated with inborn errors of metabolism (IEM). MagMet uses a highly efficient data processing procedure that performs automatic Fourier Transformation, phase correction, baseline optimization, chemical shift referencing, water signal removal, and peak picking/peak alignment. MagMet then uses the peak positions, linewidth information, and J-couplings from its own specially prepared standard metabolite reference spectral NMR library of 85 serum/plasma compounds to identify and quantify compounds from experimentally acquired NMR spectra of serum/plasma. MagMet employs linewidth adjustment for more consistent quantification of metabolites from higher field instruments and incorporates a highly efficient data processing procedure for more rapid and accurate detection and quantification of metabolites. This optimized algorithm allows the MagMet webserver to quickly detect and quantify 58 serum/plasma metabolites in 2.6 min per spectrum (when processing a dataset of 50-100 spectra). MagMet's performance was also assessed using spectra collected from defined mixtures (simulating other biofluids), with >100 previously measured plasma spectra, and from spiked serum/plasma samples simulating known IEMs. In all cases, MagMet performed with precision and accuracy matching the performance of human spectral profiling experts. MagMet is available at http://magmet.ca.

Designed Trp-Cage Proteins with Antimicrobial Activity and Enhanced Stability.

α-Helical antimicrobial peptides (αAMPs) are among the potential candidates for new anti-infectives to tackle the global crisis in antibiotic resistance, but they suffer from low bioavailability due to high susceptibility to enzymatic degradation. Here, we describe a strategy to increase the resistance of αAMPs against proteases. Fusing the 12-residue αAMP KR-12 with a Trp-cage domain induces an α-helical structure in the otherwise unfolded KR-12 moiety in solution. The resulting antimicrobial Trp-cage exhibits higher proteolytic resistance due to its stable fold as evidenced by correlating sequence-resolved digest data with structural analyses. In addition, the antimicrobial Trp-cage displays increased activity against bacteria in the presence of physiologically relevant concentrations of NaCl, while the hemolytic activity remains negligible. In contrast to previous strategies, the presented approach is not reliant on artificial amino acids and is therefore applicable to biosynthetic procedures. Our study aims to improve the pharmacokinetics of αAMPs to facilitate their use as therapeutics.

Visible-light-driven photocontrol of the Trp-cage protein fold by a diazocine cross-linker.

Diazocines are characterized by extraordinary photochemical properties rendering them of particular interest for switching the conformation of biomolecules with visible light. Current developments afford synthetic access to unprecedented diazocine derivatives promising particular opportunities in photocontrol of proteins and biological systems. In this work, the well-established approach of photocontrolling the secondary structure of α-helices was exploited using a diazocine to reversibly fold and unfold the tertiary structure of a small protein. The protein of choice was the globulary folded Trp-cage, a widely used model system for the elucidation of protein folding pathways. A specifically designed, short and rigid dicarboxy-functionalized diazocine-based cross-linker was attached to two solvent-exposed side chains at the α-helix of the miniprotein through the use of a primary amine-selective active ester. This cross-linking strategy is orthogonal to the common cysteine-based chemistry. The cross-linked Trp-cage was successfully photoisomerized and exhibited a strong correlation between protein fold and diazocine isomeric state. As determined by NMR spectroscopy, the cis-isomer stabilized the fold, while the trans-isomer led to complete protein unfolding. The successful switching of the protein fold in principle demonstrates the ability to control protein function, as the activity depends on their structural integrity.

A fast, sensitive, single-step colorimetric dipstick assay for quantifying ascorbic acid in urine.

The presence of ascorbate in human urine has been shown to be a useful dietary, fruit or vitamin C intake biomarker. More recently it has been discovered that ascorbate levels in urine can be used to facilitate the detection of precancerous colorectal polyps. While there are a number elaborate HPLC, MS or multi-step enzymatic "kit" methods to detect and quantify urinary ascorbate, these are time consuming and expensive. There are also a number of low-cost paper-based ascorbate detection dipsticks. However, the limits of detection and quantification accuracy for these dipsticks are not adequate for applications with human urine. To address these limitations, we have developed a fast, sensitive, single-step colorimetric assay that can be used to quantify ascorbate in urine and other biological fluids. The assay uses the tetrazolium salt, methylthiazolyldiphenyl-tetrazolium bromide (MTT), with the electron carrier phenazine methosulfate (PMS), in a chelated acidic phosphate-buffer to produce a vivid purple color in the presence of ascorbate. Confirmation of the performance of the assay and of its standard curve in human urine was also done using independent LC-MS/MS and NMR analyses. The lower limit of detection of the ascorbate dipstick assay described here was found to be 3.2 µM. The paper dipsticks are stable over a wide range of temperatures and can be stored for up to 150-days.

Preparation and characterization of a highly soluble Aß1-42 peptide variant.

Alzheimer's disease (AD) is a progressive neurological disease marked by the accumulation and deposition of misfolded amyloid beta or Abeta (Aß) peptide. Two species of Aß peptides are found in amyloid plaques, Aß1-40 and Aß1-42, with the latter being the more amyloidogenic of the two. Understanding how and why Aß peptides misfold, oligomerize and form amyloid plaques requires a detailed understanding of their structure and dynamics. The poor solubility and strong aggregation tendencies of Aß1-42 has made the isolation and characterization of its different structural isoforms (monomer, dimer, oligomer, amyloid) exceedingly difficult. Furthermore, while synthetic Aß1-42 peptides (Aß42syn) are readily available, the cost of isotopically labeled peptide is substantial, making their characterization by NMR spectroscopy cost prohibitive. Here we describe the design, cloning, high-level production, isotopic labeling and biophysical characterization of a modified (solubility-tagged) Aß1-42 variant that exhibits excellent water solubility and shares similar aggregation properties as wildtype Aß1-42. Specifically, we attached six lysines (6K) to the C-terminus of native Aß1-42 to create a more soluble, monomeric form of Aß1-42 called Aß42C6K. A gene for the Aß42C6K was designed, synthesized and cloned into Escherichia coli (E. coli) and the peptide was expressed at milligram levels. The Aß42C6K peptide was characterized using circular dichroism (CD), NMR, electron microscopy and thioflavin T fluorescence. Its ability to form stable monomers, oligomers and fibrils under different conditions was assessed. Our results indicate that Aß42C6K stays monomeric at high concentrations (unlike Aß1-42) and can be induced to oligomerize and form fibrils like Aß1-42. Our novel construct could be used to explore the structure and dynamics of Aß1-42 as well as the interaction of ligands with Aß1-42 via NMR.

Antitumor Anthraquinones from an Easter Island Sea Anemone: Animal or Bacterial Origin?

The presence of two known anthraquinones, Lupinacidin A and Galvaquinone B, which have antitumor activity, has been identified in the sea anemone (Gyractis sesere) from Easter Island. So far, these anthraquinones have been characterized from terrestrial and marine Actinobacteria only. In order to identify the anthraquinones producer, we isolated Actinobacteria associated with the sea anemone and obtained representatives of seven actinobacterial genera. Studies of cultures of these bacteria by HPLC, NMR, and HRLCMS analyses showed that the producer of Lupinacidin A and Galvaquinone B indeed was one of the isolated Actinobacteria. The producer strain, SN26_14.1, was identified as a representative of the genus Verrucosispora. Genome analysis supported the biosynthetic potential to the production of these compounds by this strain. This study adds Verrucosispora as a new genus to the anthraquinone producers, in addition to well-known species of Streptomyces and Micromonospora. By a cultivation-based approach, the responsibility of symbionts of a marine invertebrate for the production of complex natural products found within the animal's extracts could be demonstrated. This finding re-opens the debate about the producers of secondary metabolites in sea animals. Finally, it provides valuable information about the chemistry of bacteria harbored in the geographically-isolated and almost unstudied, Easter Island.

High-yield lithiation of azobenzenes by tin-lithium exchange.

The lithiation of halogenated azobenzenes by halogen-lithium exchange commonly leads to substantial degradation of the azo group to give hydrazine derivatives besides the desired aryl lithium species. Yields of quenching reactions with electrophiles are therefore low. This work shows that a transmetalation reaction of easily accessible stannylated azobenzenes with methyllithium leads to a near-quantitative lithiation of azobenzenes in para, meta, and ortho positions. To investigate the scope of the reaction, various lithiated azobenzenes were quenched with a variety of electrophiles. Furthermore, mechanistic (119) Sn NMR spectroscopic studies on the formation of lithiated azobenzenes are presented. A tin ate complex of the azobenzene was detected and characterized at low temperature.

Cloning and high-level expression of monomeric human superoxide dismutase 1 (SOD1) and its interaction with pyrimidine analogs.

Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or 19F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, 1H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the 15N-NMR spectra of 15N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR.

Automated Tools for the Analysis of 1D-NMR and 2D-NMR Spectra.

Nuclear magnetic resonance (NMR) spectroscopy is becoming increasingly automated. Most modern NMR spectrometers are now equipped with auto-tune/auto-match probes along with automated locking and shimming systems. Likewise, more and more instruments, especially for NMR-based metabolomics applications, are equipped with automated sample changers. All this instrumental automation allows NMR data to be collected at a rate of >100 samples/day. However, a continuing bottleneck in NMR-based metabolomics has been the time required to manually analyze and annotate the collected NMR spectra. In many cases, manual spectral annotation and analysis can take one or more hours per spectrum. Fortunately, over the past few years, several software tools have been developed that largely automate the spectral deconvolution or spectral annotation process. Using these tools requires that the samples must be prepared and the NMR spectra must be acquired in a very specific manner. In this chapter, we will describe the step-by-step preparation of biofluid samples along with the required protocols for acquiring optimal spectra for automated NMR metabolomics analysis. We will also discuss the use of three common tools (Chenomx NMR Suite, Bayesil, and COLMARm) for (semi-) automated profiling, and annotation of 1D- and 2D-NMR spectra of biofluids.

Protocols for NMR Analysis in Livestock Metabolomics.

Nuclear magnetic resonance (NMR) spectroscopy is widely considered to be one of the most robust and reproducible analytical platforms for conducting metabolomic experiments. As a metabolomic platform, NMR is not particularly sensitive, but it is nondestructive and requires no prior derivatization or chromatographic separation. It is also very automatable, easy to perform, and highly reproducible and can be used to accurately quantify dozens of metabolites in complex mixtures. To perform a successful NMR metabolomic experiment, it is important to follow good practices in sample preparation, data acquisition, and data analysis. In this chapter, we will describe, step-by-step, the preparation of different livestock samples, including both biofluids (whole blood, serum, urine, rumen content, and fecal water) and tissues (liver, muscle, testis). We will also describe the protocols for acquiring optimal NMR spectra and the techniques used to identify and quantify water-soluble metabolites by NMR spectroscopy.

Different Secondary Metabolite Profiles of Phylogenetically almost Identical Streptomyces griseus Strains Originating from Geographically Remote Locations.

As Streptomyces have shown an outstanding capacity for drug production, different campaigns in geographically distant locations currently aim to isolate new antibiotic producers. However, many of these newly isolated Streptomyces strains are classified as identical to already described species. Nevertheless, as discrepancies in terms of secondary metabolites and morphology are possible, we compared two Streptomyces strains with identical 16S rRNA gene sequences but geographically distant origins. Chosen were an Easter Island Streptomyces isolate (Streptomyces sp. SN25_8.1) and the next related type strain, which is Streptomyces griseus subsp. griseus DSM 40236T isolated from Russian garden soil. Compared traits included phylogenetic relatedness based on 16S rRNA gene sequences, macro and microscopic morphology, antibiotic activity and secondary metabolite profiles. Both Streptomyces strains shared several common features, such as morphology and core secondary metabolite production. They revealed differences in pigmentation and in the production of accessory secondary metabolites which appear to be strain-specific. In conclusion, despite identical 16S rRNA classification Streptomyces strains can present different secondary metabolite profiles and may well be valuable for consideration in processes for drug discovery.

Chemical Composition of Commercial Cow's Milk.

Bovine milk is a nutritionally rich, chemically complex biofluid consisting of hundreds of different components. While the chemical composition of cow's milk has been studied for decades, much of this information is fragmentary and very dated. In an effort to consolidate and update this information, we have applied modern, quantitative metabolomics techniques along with computer-aided literature mining to obtain the most comprehensive and up-to-date characterization of the chemical constituents in commercial cow's milk. Using nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography-mass spectrometry (LC-MS), and inductively coupled plasma-mass spectrometry (ICP-MS), we were able to identify and quantify 296 bovine milk metabolites or metabolite species (corresponding to 1447 unique structures) from a variety of commercial milk samples. Through our literature analysis, we also found another 676 metabolites or metabolite species (corresponding to 908 unique structures). Detailed information regarding all 2355 of the identified chemicals in bovine milk have been made freely available through a Web-accessible database called the Milk Composition Database or MCDB ( http://www.mcdb.ca/ ).

A Simple and Convenient Synthesis of Unlabeled and 13C-Labeled 3-(3-Hydroxyphenyl)-3-Hydroxypropionic Acid and Its Quantification in Human Urine Samples.

An improved method to synthesize the highly abundant and biomedically important urinary metabolite 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) is reported. The modified protocol is based on an indium-mediated sonochemical Reformatsky reaction. The synthesis is a simple two-step route as opposed to a complex four-step route previously reported in the literature that requires specialized equipment, flammable materials, and high-pressure reaction vessels. The described procedure also provides an expedient route to prepare a 13C isotopically labeled HPHPA that can be used as a standard for quantitative LC-MS analysis. This report also illustrates how the synthesized metabolite standard was used to detect and accurately quantify its presence in human urine samples using both NMR and LC-MS techniques.