Current Metabolomics (v.5, #1)

Meet Our Editorial Board Member by Daniel Raftery (1-2).

Preface by David G. Watson (3-3).

CASMI 2014: Challenges, Solutions and Results by Dejan Nikolic, Martin Jones, Lloyd Sumner, Warwick Dunn (5-17).
Background: The annotation or identification of small molecules during metabolomics studies is a difficult task that requires robustness in the process and reporting of the results and methods applied. The Critical Assessment of Small Molecule Identification (CASMI) contest was designed as a medium for researchers to exchange ideas and best practices related to how best to annotate and structurally identify small molecules based on mass spectrometric data.

Objective: The main objective of the CASMI contest was an unbiased evaluation of current methods for annotation and structure elucidation of small molecules.

Methods: Participants were asked to define the structures of unknown small molecules based on a set of high resolution MS and MS/MS data provided by the organizers. There were two categories to compete in: best molecular formula (category 1) and best molecular structure (category 2). A total of 42 known and 6 unknown compounds were presented as challenges. Each contestant applied their own approach to solving the challenges.

Results: Seven teams from Europe, North America and Japan participated in the contest. In category 1 three teams correctly solved 34/42 molecular formulas. In category 2 the best performing team correctly solved 24/42 structures. No correct solution in category 2 was received for unknown compounds.

Conclusion: As long as correct molecular species can be determined, determination of molecular formula from accurate mass data is reliable and robustly performed by the majority of teams. On the other hand, structure elucidation from mass spectrometric data is still a challenging task, particularly for true unknowns.


Background: Identification of detected compounds in untargeted LC/MS profiling is a common bottleneck in metabolomics. The CASMI contest challenges mass spectrometry experts and algorithm developers to evaluate how reliable their methods derive molecular formulae and structures from blinded mass spectral data.

Objective: The application of the MAGMa software to solve the CASMI 2014 challenges is described.

Methods: MAGMa was used to automatically retrieve candidate molecular structures from the HMDB and PubChem chemical databases, based on MS1 precursor m/z values, and to provide a score indicating how well they explain the accurate MS2 spectra.

Results: For 40 out of 48 challenges, candidates with the correct molecular formula were ranked on top. For 22 out of 42 challenges the top-ranked candidate also represented the correct chemical structure and in 9 other cases the correct molecule was ranked in the top 10.

Conclusion: Advantages and limitations of the approach and consequences with respect to retrieving and scoring of the correct candidates are discussed.


Background: Automated annotation of MS spectra remains highly challenging. Therefore, the Critical Assessment of Small Molecule Identification (CASMI) Contest (http://casmi-contest.org/) represents a unique opportunity to blindly evaluate an annotation workflow.

Objective: The 2014 CASMI Contest consisted of 42 MS and MS2 spectra (from “molecules detected in mammalian biofluids and tissues”, “natural products of plant, fungal or bacterial origin”, “synthetic or semi-synthetic molecules”), which had to be identified (molecular formulae and structures).

Methods: An R script based on the Rdisop and RMassBank packages was devised for the automated annotation of the provided spectra. Searching within various databases yielded the assignment of structures. Further discrimination between annotations was achieved using phylogenetic information and in silico fragmentation by competitive fragmentation modelling using CFM-ID.

Results: Successes and failures of the proposed script were investigated after release of the CASMI Contest solutions.

Conclusion: The CASMI Contest allowed identifying key points for the successful annotation of MS spectra. As an example, this study pointed out the importance of taking a large number of adducts into consideration (such as [M+H]+, [M+H-H2O]+, [M+Na]+, [M+K]+ and [M]+ in positive ionization) to avoid failed annotations.


CFM-ID Applied to CASMI 2014 by Felicity Allen, Russell Greiner, David Wishart (35-39).
We describe our winning submission to the Critical Assessment of Small Molecule Identification (CASMI) 2014 competition, based on our method called Competitive Fragmentation Modeling (CFM-ID).

Background: The CASMI competition is an annual international competition in which contestants must provide putative chemical structures for each of a set of challenge mass spectra.

Methods: For each target spectrum, we used CFM-ID to provide a score for each candidate compound, based on its ESI-MS/MS spectrum. We combined this with two other scoring components that made use of additional meta information provided with each challenge. We also investigated the role of this meta information in the results obtained.

Results: The resulting method correctly identified 24 out of 42 challenge compounds, and ranked the correct candidate in the top 10 in 33 challenges. The use of the meta information was found to play a significant role in the identification performance.

Conclusion: This was sufficient to win the CASMI competition, which is evolving to become an important arena for assessment of progress in this field.


Background: The CASMI 2014 contest presents challenges in structure determination using mass spectrometry data.

Objective: The contestants solve the problems comparing different software approaches.

Methods: This paper reports a manual approach to structure determination.

Results: The author successfully determined 31/42 molecular formulas and 21/42 structures. Five of the formulas were identified correctly but reported incorrectly. In 4 cases the author identified the wrong ion as the molecular species and so calculated the formula of an adduct or a fragment. In two cases, the author misidentified the formula due to not considering all possible atom combinations. For 6 of the structure misses, the author started with the wrong formula. In 13 cases, the author had the correct formula but reported only one of the possible isomers and these were the wrong isomer. In two cases, no result was reported.

Conclusion: The manual approach is successful at obtaining the correct molecular formula but made it more difficult to generate a list of possible isomers. Suggestions are made for including relative retention, pH effect on retention and H/D exchange information in future CASMI challenges to facilitate isomer identification.


Reproducibility and Stability of Aqueous Metabolite Levels in Extracted Serum by NMR Spectroscopy by Matthew M. Miele, Brian A. Irving, Broc R. Wenrich, Phillip L. Martin, David Rovnyak (45-54).
Background: Metabolomics offers the potential of correlating a macroscopic view of an organism to measured levels of small molecule reporters of metabolic pathways. Despite strong growth in metabolomics studies, questions on reproducibility and sample stability deserve a closer look.

Objective: This work measured acetonitrile extractions of the aqueous components of fetal bovine serum (FBS) by 1H NMR spectroscopy to determine the stability and reproducibility of metabolite levels over time at storage temperatures of 20, 4, -30, and -80 °C.

Method: First, mock sera, spiked sera, and pooled human sera were used to find the measurement precision and detection limits of the instrumentation used here (600 MHz, roomtemperature triple resonance probe). Next, using four replicates at each of four storage temperatures, 48 metabolites extracted (2:1 acetonitrile to serum) from FBS samples were profiled over several time scales.

Results: Although most metabolites were found to be more stable than expected at room temperature, ca. two weeks, allantoin, creatinine, and glutamine degraded much more rapidly than others at both room temperature and 4 ?C, measurably decreasing over a few hours or 1 day, respectively. Storing samples at 4 °C dramatically improves the lifetime of all metabolites, while the fidelity of extracted samples over very long term storage at -30 and -80 ?C is supported by this work. Slight degradation of the cryogenically stored serum extracts is linked to freeze-thaw cycles.

Conclusion: The poor stability of a few metabolites for short times supports vigilance in minimizing and standardizing room temperature handling and refrigeration of extracted samples, as inconsistent sample storage even on short time scales would introduce variation that would confound clustering.


Serum Metabolic Disturbances Hailing in Initial Hours of Acute Myocardial Infarction Elucidated by NMR based Metabolomics by Atul Rawat, Rohit K. Srivastava, Durgesh Dubey, Anupam Guleria, Sanjay Singh, Anand Prakash, Dinesh R. Modi, C. L. Khetrapal, Sunita Tiwari, Dinesh Kumar (55-67).
Background: Acute myocardial infarction (AMI) is a serious medical emergency and leading cause of cardiac-related deaths worldwide. The devastating outcome is sudden death of the patient within first few hours from the onset of symptoms. The rapid detection of physiological transformations associated with AMI coupled with instant treatment to reset these changes and monitoring response to treatment can greatly decrease the mortality and morbidity of patients.

Objective: To establish the early hour metabolomic signatures in the sera of AMI patieints.

Methodology: Metabolic profiles of sera collected from 42 AMI patients (immediately after the myocardial infarction) and 38 age/sex matched normal controls were obtained using high-resolution 1D 1H CPMG and diffusion edited NMR spectra. The metabolic profiles were compared using multivariate statistical analysis to identify the disease specific metabolic disturbances associated with AMI and, therefore, the perturbed biochemical pathways in this condition.

Results: Our results revealed significant metabolic perturbations in AMI compared to control cohorts. The upregulated metabolites in AMI condition include arginine, glycine, tyrosine, phenylalanine, glucose, creatine, creatinine, lactate, N-acetyl glycoproteins and phospholipids, while the levels of amino acids (such as valine, alanine, glutamate, glutamine, threonine and methionine), citrate, acetone, choline, glycero-phosphocholine, trimethylamine-N-oxide and lipids/fatty acids were decreased. Receiver operating curve characteristics (ROC) confirmed the robustness and validity of these metabolic markers.

Conclusion: The resulted metabolic profiling provided new insights into the metabolic processes involved in acute myocardial infarction. Further, we foresee that these changes would aid rapid clinical evaluation of myocardial infarction in emergency and its timely management.


The Metabolic Response of Glycogen and Free Fatty Acids to Endurance Exercise by Piero L. Ipata, Francesco Balestri, Rossana Pesi (68-75).
Background: To date the metabolic response to physical exercise in humans is a topic of great interest, in view of the importance of sports in modern society. Oxygen-independent glycogenolysis is satisfactorily reported in textbooks and in specialized reviews as a paradigm the metabolic response to intense muscle contraction, as it occurs in the 100 meter sprint.

Focus: Here we propose to carefully analyze the more complex oxygen-dependent glycogen and free fatty acids utilization in sustained contracting muscle, as it occurs in the marathon endurance race. The reactions of aerobic glycogenolysis, tricarboxyilic acid cycle, and free fatty acids catabolism, although well known long time ago, are presened in a systematic manner, to facilitate understanding of their central role in ATP generation. The metabolic response of glycogen to intense muscle exercise mainly consites in the exergonic oxygen-independent conversion of each glycosyl residue into two pyruvate molecules, with the concomitant production of ATP, the substrate of mysosin ATPase. The two pyruvate molecule enter the Tricaboxylic Acid Cycle, and generate additional ATP molecules. Particular emphasis is given to the metabolic response of fatty acids derived from lipid stores to long distance endurance race.

Prospects: Memorizing a long series of reactions might appear a boring task. However, a long didactic experience convinced one of us (P. L. I.) that discussing through a guided approach the detailed metabolic networks responsible for the synthesis of ATP, the substrate of myosin ATPase, is as rewarding as memorizing the various physiological steps of muscle contraction. Finally, a deep knowledge of the metabolomics of oxygen-dependent and oxigen-independent muscle contration will greatly help students to get a clear idea of two important muscle physiological concepts: muscle capacity and muscle power.