Optimized Analytical Procedures for the Untargeted Metabolomic Profiling of Human Urine and Plasma by Combining Hydrophilic Interaction (HILIC) and Reverse-Phase Liquid Chromatography (RPLC)-Mass Spectrometry.

Profiling of body fluids is crucial for monitoring and discovering metabolic markers of health and disease and for providing insights into human physiology. Since human urine and plasma each contain an extreme diversity of metabolites, a single liquid chromatographic system when coupled to mass spectrometry (MS) is not sufficient to achieve reasonable metabolome coverage. Hydrophilic interaction liquid chromatography (HILIC) offers complementary information to reverse-phase liquid chromatography (RPLC) by retaining polar metabolites. With the objective of finding the optimal combined chromatographic solution to profile urine and plasma, we systematically investigated the performance of five HILIC columns with different chemistries operated at three different pH (acidic, neutral, basic) and five C18-silica RPLC columns. The zwitterionic column ZIC-HILIC operated at neutral pH provided optimal performance on a large set of hydrophilic metabolites. The RPLC columns Hypersil GOLD and Zorbax SB aq were proven to be best suited for the metabolic profiling of urine and plasma, respectively. Importantly, the optimized HILIC-MS method showed excellent intrabatch peak area reproducibility (CV < 12%) and good long-term interbatch (40 days) peak area reproducibility (CV < 22%) that were similar to those of RPLC-MS procedures. Finally, combining the optimal HILIC- and RPLC-MS approaches greatly expanded metabolome coverage with 44% and 108% new metabolic features detected compared with RPLC-MS alone for urine and plasma, respectively. The proposed combined LC-MS approaches improve the comprehensiveness of global metabolic profiling of body fluids and thus are valuable for monitoring and discovering metabolic changes associated with health and disease in clinical research studies

Towards quantitative mass spectrometry based metabolomics in microbial and mammalian systems

Metabolome analyses are a suite of analytical approaches that enable us to capture changes in the metabolome (small molecular weight components, typically less than 1500 Da) in biological systems. Mass spectrometry (MS) has been widely used for this purpose. The key challenge here is to be able to capture changes in a reproducible and reliant manner that is representative of the events that take place in vivo. Typically, the analysis is carried out in vitro, by isolating the system and extracting the metabolome. MS-based approaches enable us to capture metabolomic changes with high sensitivity and resolution. When developing the technique for different biological systems, there are similarities in challenges and differences that are specific to the system under investigation. Here, we review some of the challenges in capturing quantitative changes in the metabolome with MS based approaches, primarily in microbial and mammalian systems

Updates in metabolomics tools and resources: 2014-2015

Data processing and interpretation represent the most challenging and time-consuming steps in high-throughput metabolomic experiments, regardless of the analytical platforms (MS or NMR spectroscopy based) used for data acquisition. Improved machinery in metabolomics generates increasingly complex datasets that create the need for more and better processing and analysis software and in silico approaches to understand the resulting data. However, a comprehensive source of information describing the utility of the most recently developed and released metabolomics resources—in the form of tools, software, and databases—is currently lacking. Thus, here we provide an overview of freely-available, and open-source, tools, algorithms, and frameworks to make both upcoming and established metabolomics researchers aware of the recent developments in an attempt to advance and facilitate data processing workflows in their metabolomics research. The major topics include tools and researches for data processing, data annotation, and data visualization in MS and NMR-based metabolomics. Most in this review described tools are dedicated to untargeted metabolomics workflows; however, some more specialist tools are described as well. All tools and resources described including their analytical and computational platform dependencies are summarized in an overview Table

Statistical Workflow for Feature Selection in Human Metabolomics Data.

High-throughput metabolomics investigations, when conducted in large human cohorts, represent a potentially powerful tool for elucidating the biochemical diversity underlying human health and disease. Large-scale metabolomics data sources, generated using either targeted or nontargeted platforms, are becoming more common. Appropriate statistical analysis of these complex high-dimensional data will be critical for extracting meaningful results from such large-scale human metabolomics studies. Therefore, we consider the statistical analytical approaches that have been employed in prior human metabolomics studies. Based on the lessons learned and collective experience to date in the field, we offer a step-by-step framework for pursuing statistical analyses of cohort-based human metabolomics data, with a focus on feature selection. We discuss the range of options and approaches that may be employed at each stage of data management, analysis, and interpretation and offer guidance on the analytical decisions that need to be considered over the course of implementing a data analysis workflow. Certain pervasive analytical challenges facing the field warrant ongoing focused research. Addressing these challenges, particularly those related to analyzing human metabolomics data, will allow for more standardization of as well as advances in how research in the field is practiced. In turn, such major analytical advances will lead to substantial improvements in the overall contributions of human metabolomics investigations

MetaboAnalystR 3.0: Toward an Optimized Workflow for Global Metabolomics.

Liquid chromatography coupled to high-resolution mass spectrometry platforms are increasingly employed to comprehensively measure metabolome changes in systems biology and complex diseases. Over the past decade, several powerful computational pipelines have been developed for spectral processing, annotation, and analysis. However, significant obstacles remain with regard to parameter settings, computational efficiencies, batch effects, and functional interpretations. Here, we introduce MetaboAnalystR 3.0, a significantly improved pipeline with three key new features: (1) efficient parameter optimization for peak picking; (2) automated batch effect correction; and 3) more accurate pathway activity prediction. Our benchmark studies showed that this workflow was 20~100X faster compared to other well-established workflows and produced more biologically meaningful results. In summary, MetaboAnalystR 3.0 offers an efficient pipeline to support high-throughput global metabolomics in the open-source R environment

Multivariate Analysis in Metabolomics

Metabolomics aims to provide a global snapshot of all small-molecule metabolites in cells and biological fluids, free of observational biases inherent to more focused studies of metabolism. However, the staggeringly high information content of such global analyses introduces a challenge of its own; efficiently forming biologically relevant conclusions from any given metabolomics dataset indeed requires specialized forms of data analysis. One approach to finding meaning in metabolomics datasets involves multivariate analysis (MVA) methods such as principal component analysis (PCA) and partial least squares projection to latent structures (PLS), where spectral features contributing most to variation or separation are identified for further analysis. However, as with any mathematical treatment, these methods are not a panacea; this review discusses the use of multivariate analysis for metabolomics, as well as common pitfalls and misconceptions

Multivariate Analysis in Metabolomics

Metabolomics aims to provide a global snapshot of all small-molecule metabolites in cells and biological fluids, free of observational biases inherent to more focused studies of metabolism. However, the staggeringly high information content of such global analyses introduces a challenge of its own; efficiently forming biologically relevant conclusions from any given metabolomics dataset indeed requires specialized forms of data analysis. One approach to finding meaning in metabolomics datasets involves multivariate analysis (MVA) methods such as principal component analysis (PCA) and partial least squares projection to latent structures (PLS), where spectral features contributing most to variation or separation are identified for further analysis. However, as with any mathematical treatment, these methods are not a panacea; this review discusses the use of multivariate analysis for metabolomics, as well as common pitfalls and misconceptions

Comparison of HPLC and NMR for quantification of the main volatile fatty acids in rumen digesta

Accurate quantification of volatile fatty acid (VFA) concentrations in rumen fluid are essential for research on rumen metabolism. The study comprehensively investigated the pros and cons of High-performance liquid chromatography (HPLC) and (1)H Nuclear magnetic resonance ((1)H-NMR) analysis methods for rumen VFAs quantification. We also investigated the performance of several commonly used data pre-treatments for the two sets of data using correlation analysis, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The molar proportion and reliability analysis demonstrated that the two approaches produce highly consistent VFA concentrations. In the pre-processing of NMR spectra, line broadening and shim correction may reduce estimated concentrations of metabolites. We observed differences in results using multiplet of different protons from one compound and identified “handle signals” that provided the most consistent concentrations. Different data pre-treatment strategies tested with both HPLC and NMR significantly affected the results of downstream data analysis. “Normalized by sum” pre-treatment can eliminate a large number of positive correlations between NMR-based VFA. A “Combine” strategy should be the first choice when calculating the correlation between metabolites or between samples. The PCA and PLS-DA suggest that except for “Normalize by sum”, pre-treatments should be used with caution