Assessment of Metabolome Annotation Quality: A Method for Evaluating the False Discovery Rate of Elemental Composition Searches

Background

In metabolomics researches using mass spectrometry (MS), systematic searching of high-resolution mass data against compound databases is often the first step of metabolite annotation to determine elemental compositions possessing similar theoretical mass numbers. However, incorrect hits derived from errors in mass analyses will be included in the results of elemental composition searches. To assess the quality of peak annotation information, a novel methodology for false discovery rates (FDR) evaluation is presented in this study. Based on the FDR analyses, several aspects of an elemental composition search, including setting a threshold, estimating FDR, and the types of elemental composition databases most reliable for searching are discussed.

Methodology/Principal Findings

The FDR can be determined from one measured value (i.e., the hit rate for search queries) and four parameters determined by Monte Carlo simulation. The results indicate that relatively high FDR values (30–50%) were obtained when searching time-of-flight (TOF)/MS data using the KNApSAcK and KEGG databases. In addition, searches against large all-in-one databases (e.g., PubChem) always produced unacceptable results (FDR >70%). The estimated FDRs suggest that the quality of search results can be improved not only by performing more accurate mass analysis but also by modifying the properties of the compound database. A theoretical analysis indicates that FDR could be improved by using compound database with smaller but higher completeness entries.

Conclusions/Significance

High accuracy mass analysis, such as Fourier transform (FT)-MS, is needed for reliable annotation (FDR <10%). In addition, a small, customized compound database is preferable for high-quality annotation of metabolome data.     

Metabolic Alterations in Two Cirsium Species Identified at Distinct Phenological Stages using UPLC‐QTOF/MS

Introduction

Cirsium chanroenicum and C. setidens are commonly used both in traditional folk medicine and as a food source. The quality of different species of Cirsium at different harvest times is a function of their metabolite composition, which is determined by the phenological stage.

Objective

We sought to determine the differences in the metabolite composition of two species of Cirsium during different phenological stages using ultra‐performance liquid chromatography (UPLC) quadrupole time‐of‐flight (QTOF) mass spectrometry (MS).

Methodology

Cirsium chanroenicum and C. setidens plants were collected at the floral budding and full flowering stages. Metabolic profiles of Cirsium extracts were determined using UPLC‐QTOF/MS to characterise the differences between phenological stages, and the major metabolites were quantified using UPLC‐QTOF/MS‐multiple reaction monitoring (MRM).

Results

At the full flowering stage, the levels of phenolic acids as well as components of the phenylpropanoid pathway were increased. Flavonoids predominated at the full flowering stage in both species. The levels of coumaric acid, kaempferol, and pectolinarigenin differed between the two species of Cirsium. Overall, these results suggest that components of the phenylpropanoid metabolic pathway are upregulated in the full flowering stage in Cirsium, although we did observe some variation between the species.

Conclusion

These results will help elucidate the metabolic pathways related to the different phases of the vegetative cycle, and may help determine the optimal season for the harvest of Cirsium with the highest levels of bioactive compounds. Copyright © 2017 John Wiley & Sons, Ltd.     

Metabolomic database annotations via query of elemental compositions: Mass accuracy is insufficient even at less than 1 ppm

Background  

Metabolomic studies are targeted at identifying and quantifying all metabolites in a given biological context. Among the tools used for metabolomic research, mass spectrometry is one of the most powerful tools. However, metabolomics by mass spectrometry always reveals a high number of unknown compounds which complicate in depth mechanistic or biochemical understanding. In principle, mass spectrometry can be utilized within strategies of de novo structure elucidation of small molecules, starting with the computation of the elemental composition of an unknown metabolite using accurate masses with errors <5 ppm (parts per million). However even with very high mass accuracy (<1 ppm) many chemically possible formulae are obtained in higher mass regions. In automatic routines an additional orthogonal filter therefore needs to be applied in order to reduce the number of potential elemental compositions. This report demonstrates the necessity of isotope abundance information by mathematical confirmation of the concept.     

HybGFS: a hybrid method for genome-fingerprint scanning

Background  

Protein identification based on mass spectrometry (MS) has previously been performed using peptide mass fingerprinting (PMF) or tandem MS (MS/MS) database searching. However, these methods cannot identify proteins that are not already listed in existing databases. Moreover, the alternative approach of de novo sequencing requires costly equipment and the interpretation of complex MS/MS spectra. Thus, there is a need for novel high-throughput protein-identification methods that are independent of existing predefined protein databases.     

Proteomic study on gender differences in aging kidney of mice

Background  

This study aims to analyze sex differences in mice aging kidney. We applied a proteomic technique based on subfractionation, and liquid chromatography coupled with 2-DE. Samples from male and female CD1-Swiss outbred mice from 28 weeks, 52 weeks, and 76 weeks were analysed by 2-DE, and selected proteins were identified by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS).     

The Neural/Immune Gene Ontology: clipping the Gene Ontology for neurological and immunological systems

Background  

The Gene Ontology (GO) is used to describe genes and gene products from many organisms. When used for functional annotation of microarray data, GO is often slimmed by editing so that only higher level terms remain. This practice is designed to improve the summarizing of experimental results by grouping high level terms and the statistical power of GO term enrichment analysis.     

The Spatial Distribution of Alkaloids in Psychotria prunifolia (Kunth) Steyerm and Palicourea coriacea (Cham.) K. Schum Leaves Analysed by Desorption Electrospray Ionisation Mass Spectrometry Imaging

Introduction

Species of the genera Psychotria and Palicourea are sources of indole alkaloids, however, the distribution of alkaloids within the plants is not known. Analysing the spatial distribution using desorption electrospray ionisation mass spectrometry imaging (DESI‐MSI) has become attractive due to its simplicity and high selectivity compared to traditional histochemical techniques.

Objectives

To apply DESI‐MSI to visualise the alkaloid distribution on the leaf surface of Psychotria prunifolia and Palicourea coriacea and to compare the distributions with HPLC–MS and histochemical analyses.

Methodology

Based upon previous structure elucidation studies, four alkaloids targeted in this study were identified using high resolution mass spectrometry by direct infusion of plant extracts, and their distributions were imaged by DESI‐MSI via tissue imprints on a porous Teflon surface. Relative quantitation of the four alkaloids was obtained by HPLC–MS/MS analysis performed using multiple‐reaction monitoring (MRM) mode on a triple quadrupole mass spectrometer.

Results

Alkaloids showed distinct distributions on the leaf surfaces. Prunifoleine was mainly present in the midrib, while 10‐hydroxyisodeppeaninol was concentrated close to the petiole; a uniform distribution of 10‐hydroxyantirhine was observed in the whole leaf of Psychotria prunifolia. The imprinted image from the Palicourea coriacea leaf also showed a homogeneous distribution of calycanthine throughout the leaf surface.

Conclusion

Different distributions were found for three alkaloids in Psychotria prunifolia, and the distributions found by MSI were in complete accordance with HPLC–MS analysis and histochemical results. The DESI‐MSI technique was therefore demonstrated to provide reliable information about the spatial distribution of metabolites in plants. Copyright © 2017 John Wiley & Sons, Ltd.     

Development and experimental verification of a genome-scale metabolic model for Corynebacterium glutamicum

Background  

In silico genome-scale metabolic models enable the analysis of the characteristics of metabolic systems of organisms. In this study, we reconstructed a genome-scale metabolic model of Corynebacterium glutamicum on the basis of genome sequence annotation and physiological data. The metabolic characteristics were analyzed using flux balance analysis (FBA), and the results of FBA were validated using data from culture experiments performed at different oxygen uptake rates.     

multiplierz: an extensible API based desktop environment for proteomics data analysis

Background  

Efficient analysis of results from mass spectrometry-based proteomics experiments requires access to disparate data types, including native mass spectrometry files, output from algorithms that assign peptide sequence to MS/MS spectra, and annotation for proteins and pathways from various database sources. Moreover, proteomics technologies and experimental methods are not yet standardized; hence a high degree of flexibility is necessary for efficient support of high- and low-throughput data analytic tasks. Development of a desktop environment that is sufficiently robust for deployment in data analytic pipelines, and simultaneously supports customization for programmers and non-programmers alike, has proven to be a significant challenge.     

Quaternary structure of <Emphasis Type="Italic">Artemia</Emphasis> haemoglobin II: analysis of T and C polymer alignment and interpolymer interface

Background  

The brine shrimp Artemia expresses four different types of haemoglobin subunits namely C1, C2, T1 and T2. Two of these four subunits dimerize in different combinations to produce the three isoforms of the heterodimeric Artemia haemoglobin: HbI (C1 and C2), HbII (C1 and T2) and HbIII (T1 and T2). Previous biochemical, biophysical and computational analyses demonstrate that the T and C polymers are rings of nine concatenated globin domains, which are covalently joined by interdomain linkers. Two such rings stacked coaxially give the functional molecule. This research aimed to construct a quaternary structural model of Artemia HbII that shows the interpolymer interface and domain-domain alignment, using the MS3D (mass spectrometry for three dimensional analysis) approach. This involved introducing chemical crosslinks between the two polymers, cleaving with trypsin and analyzing the resulting products by mass spectrometry. This was followed by computational analysis of the mass spectrometry data using the program SearchXlinks to identify putatively crosslinked peptides.     

A compatible exon-exon junction database for the identification of exon skipping events using tandem mass spectrum data

Background  

Alternative splicing is an important gene regulation mechanism. It is estimated that about 74% of multi-exon human genes have alternative splicing. High throughput tandem (MS/MS) mass spectrometry provides valuable information for rapidly identifying potentially novel alternatively-spliced protein products from experimental datasets. However, the ability to identify alternative splicing events through tandem mass spectrometry depends on the database against which the spectra are searched.     

The zebra finch neuropeptidome: prediction,detection and expression

Background  

Among songbirds, the zebra finch (Taeniopygia guttata) is an excellent model system for investigating the neural mechanisms underlying complex behaviours such as vocal communication, learning and social interactions. Neuropeptides and peptide hormones are cell-to-cell signalling molecules known to mediate similar behaviours in other animals. However, in the zebra finch, this information is limited. With the newly-released zebra finch genome as a foundation, we combined bioinformatics, mass-spectrometry (MS)-enabled peptidomics and molecular techniques to identify the complete suite of neuropeptide prohormones and final peptide products and their distributions.     

Toward better annotation in plant metabolomics: isolation and structure elucidation of 36 specialized metabolites from Oryza sativa (rice) by using MS/MS and NMR analyses

Metabolomics plays an important role in phytochemical genomics and crop breeding; however, metabolite annotation is a significant bottleneck in metabolomic studies. In particular, in liquid chromatography–mass spectrometry (MS)-based metabolomics, which has become a routine technology for the profiling of plant-specialized metabolites, a substantial number of metabolites detected as MS peaks are still not assigned properly to a single metabolite. Oryza sativa (rice) is one of the most important staple crops in the world. In the present study, we isolated and elucidated the structures of specialized metabolites from rice by using MS/MS and NMR. Thirty-six compounds, including five new flavonoids and eight rare flavonolignan isomers, were isolated from the rice leaves. The MS/MS spectral data of the isolated compounds, with a detailed interpretation of MS fragmentation data, will facilitate metabolite annotation of the related phytochemicals by enriching the public mass spectral data depositories, including the plant-specific MS/MS-based database, ReSpect.     

Unbiased characterization of genotype-dependent metabolic regulations by metabolomic approach in Arabidopsis thaliana

Background  

Metabolites are not only the catalytic products of enzymatic reactions but also the active regulators or the ultimate phenotype of metabolic homeostasis in highly complex cellular processes. The modes of regulation at the metabolome level can be revealed by metabolic networks. We investigated the metabolic network between wild-type and 2 mutant (methionine-over accumulation 1 [mto1] and transparent testa4 [tt4]) plants regarding the alteration of metabolite accumulation in Arabidopsis thaliana.     

Transformation and other factors of the peptide mass spectrometry pairwise peak-list comparison process

Background:  

Biological Mass Spectrometry is used to analyse peptides and proteins. A mass spectrum generates a list of measured mass to charge ratios and intensities of ionised peptides, which is called a peak-list. In order to classify the underlying amino acid sequence, the acquired spectra are usually compared with synthetic ones. Development of suitable methods of direct peak-list comparison may be advantageous for many applications.     

Proteome of the phytopathogen <Emphasis Type="Italic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis>: a global expression profile

Background  

Citrus canker is a disease caused by Xantomonas citri subsp.citri (Xac), and has emerged as one of the major threats to the worldwide citrus crop because it affects all commercial citrus varieties, decreases the production and quality of the fruits and can spread rapidly in citrus growing areas. In this work, the first proteome of Xac was analyzed using two methodologies, two-dimensional liquid chromatography (2D LC) and tandem mass spectrometry (MS/MS).     

A new measure for functional similarity of gene products based on Gene Ontology

Background  

Gene Ontology (GO) is a standard vocabulary of functional terms and allows for coherent annotation of gene products. These annotations provide a basis for new methods that compare gene products regarding their molecular function and biological role.