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1.
Nat Methods ; 20(2): 193-204, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36543939

RESUMO

Progress in mass spectrometry lipidomics has led to a rapid proliferation of studies across biology and biomedicine. These generate extremely large raw datasets requiring sophisticated solutions to support automated data processing. To address this, numerous software tools have been developed and tailored for specific tasks. However, for researchers, deciding which approach best suits their application relies on ad hoc testing, which is inefficient and time consuming. Here we first review the data processing pipeline, summarizing the scope of available tools. Next, to support researchers, LIPID MAPS provides an interactive online portal listing open-access tools with a graphical user interface. This guides users towards appropriate solutions within major areas in data processing, including (1) lipid-oriented databases, (2) mass spectrometry data repositories, (3) analysis of targeted lipidomics datasets, (4) lipid identification and (5) quantification from untargeted lipidomics datasets, (6) statistical analysis and visualization, and (7) data integration solutions. Detailed descriptions of functions and requirements are provided to guide customized data analysis workflows.


Assuntos
Biologia Computacional , Lipidômica , Biologia Computacional/métodos , Software , Informática , Lipídeos/química
2.
Anal Chem ; 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-39028917

RESUMO

The analysis of gangliosides is extremely challenging, given their structural complexity, lack of reference standards, databases, and software solutions. Here, we introduce a fast 6 min high field asymmetric ion mobility spectrometry (FAIMS) shotgun lipidomics workflow, along with a dedicated software solution for ganglioside detection. By ramping FAIMS compensation voltages, ideal ranges for different ganglioside classes were obtained. FAIMS revealed both class- and charge-state separation behavior based on the glycan headgroup moiety. The number of sialic acids attached to the glycan moiety correlates positively with their preferred charge states, i.e., trisialylated gangliosides were mainly present as [M - 3H]3- ions, whereas [M - 4H]4- and [M - 5H]5- ions were observed for GQ1 and GP1. For data evaluation, we developed a shotgun/FAIMS extension for the open-source Lipid Data Analyzer (LDA), enabling automated annotation of gangliosides up to the molecular lipid species level. This extension utilized combined orthogonal fragmentation spectra from CID, HCD, and 213 nm UVPD ion activation methods and covers 29 ganglioside classes, including acetylated and fucosylated modifications. With our new workflow and software extension 117 unique gangliosides species were identified in porcine brain extracts. While conventional shotgun lipidomics favored the observation of singly charged ganglioside species, the utilization of FAIMS made multiply charged lipid species accessible, resulting in an increased number of detected species, primarily due to an improved signal-to-noise ratio arising from FAIMS charge state filtering. Therefore, this FAIMS-driven workflow, complemented by new software capabilities, offers a promising strategy for complex ganglioside and glycosphingolipid characterization in shotgun lipidomics.

3.
J Lipid Res ; 62: 100104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34384788

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is a common metabolic dysfunction leading to hepatic steatosis. However, NAFLD's global impact on the liver lipidome is poorly understood. Using high-resolution shotgun mass spectrometry, we quantified the molar abundance of 316 species from 22 major lipid classes in liver biopsies of 365 patients, including nonsteatotic patients with normal or excessive weight, patients diagnosed with NAFL (nonalcoholic fatty liver) or NASH (nonalcoholic steatohepatitis), and patients bearing common mutations of NAFLD-related protein factors. We confirmed the progressive accumulation of di- and triacylglycerols and cholesteryl esters in the liver of NAFL and NASH patients, while the bulk composition of glycerophospho- and sphingolipids remained unchanged. Further stratification by biclustering analysis identified sphingomyelin species comprising n24:2 fatty acid moieties as membrane lipid markers of NAFLD. Normalized relative abundance of sphingomyelins SM 43:3;2 and SM 43:1;2 containing n24:2 and n24:0 fatty acid moieties, respectively, showed opposite trends during NAFLD progression and distinguished NAFL and NASH lipidomes from the lipidome of nonsteatotic livers. Together with several glycerophospholipids containing a C22:6 fatty acid moiety, these lipids serve as markers of early and advanced stages of NAFL.


Assuntos
Lipidômica , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Metabolismo dos Lipídeos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
4.
Anal Chem ; 92(20): 14054-14062, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33003696

RESUMO

Sphingolipids constitute a heterogeneous lipid category that is involved in many key cellular functions. For high-throughput analyses of sphingolipids, tandem mass spectrometry (MS/MS) is the method of choice, offering sufficient sensitivity, structural information, and quantitative precision for detecting hundreds to thousands of species simultaneously. While glycerolipids and phospholipids are predominantly non-hydroxylated, sphingolipids are typically dihydroxylated. However, species containing one or three hydroxylation sites can be detected frequently. This variability in the number of hydroxylation sites on the sphingolipid long-chain base and the fatty acyl moiety produces many more isobaric species and fragments than for other lipid categories. Due to this complexity, the automated annotation of sphingolipid species is challenging, and incorrect annotations are common. In this study, we present an extension of the Lipid Data Analyzer (LDA) "decision rule set" concept that considers the structural characteristics that are specific for this lipid category. To address the challenges inherent to automated annotation of sphingolipid structures from MS/MS data, we first developed decision rule sets using spectra from authentic standards and then tested the applicability on biological samples including murine brain and human plasma. A benchmark test based on the murine brain samples revealed a highly improved annotation quality as measured by sensitivity and reliability. The results of this benchmark test combined with the easy extensibility of the software to other (sphingo)lipid classes and the capability to detect and correctly annotate novel sphingolipid species make LDA broadly applicable to automated sphingolipid analysis, especially in high-throughput settings.


Assuntos
Encéfalo/metabolismo , Sistemas Computadorizados de Registros Médicos/instrumentação , Plasma/metabolismo , Esfingolipídeos/análise , Esfingolipídeos/metabolismo , Animais , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Ácidos Graxos/química , Ensaios de Triagem em Larga Escala , Humanos , Hidroxilação , Camundongos , Modelos Químicos , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
5.
Nat Methods ; 14(12): 1171-1174, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29058722

RESUMO

We achieve automated and reliable annotation of lipid species and their molecular structures in high-throughput data from chromatography-coupled tandem mass spectrometry using decision rule sets embedded in Lipid Data Analyzer (LDA; http://genome.tugraz.at/lda2). Using various low- and high-resolution mass spectrometry instruments with several collision energies, we proved the method's platform independence. We propose that the software's reliability, flexibility, and ability to identify novel lipid molecular species may now render current state-of-the-art lipid libraries obsolete.


Assuntos
Cromatografia Líquida/métodos , Lipídeos/análise , Lipídeos/química , Espectrometria de Massas em Tandem/métodos , Algoritmos , Animais , Fígado/química , Camundongos , Estrutura Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
6.
Anal Chem ; 91(20): 12615-12618, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31525911

RESUMO

mzTab 2.0 for metabolomics (mzTab-M) is the most recent standard format developed in collaboration by the Proteomics and Metabolomics Standards Initiatives including contributions by the recently founded Lipidomics Standards Initiative. mzTab-M is a redesign of the original mzTab format which was geared toward reporting of proteomics results and, as such, provided only limited support for metabolites. As a tab-delimited, spreadsheet-like format, mzTab-M captures experimental metadata, summary information on small molecules across assays, MS features as a basis for quantitation, and evidence to support the reporting of individual or feature group identifications. Here, we present the Java reference implementation for reading, writing, and validating mzTab-M files. Furthermore, we provide a web application for conveniently validating mzTab-M files by a graphical user interface, and a command line validator that accompanies the library. The jmzTab-M library, version 1.0.4 ( https://doi.org/10.5281/zenodo.3362151 ), is available at https://github.com/lifs-tools/jmzTab-m and from Maven Central at https://search.maven.org/search?q=jmztabm under the terms of the open source Apache License 2.0. The web application as well as the Python and R implementations are available at https://github.com/lifs-tools . The respective Web sites link to additional API documentation, as well as to usage examples.


Assuntos
Metabolômica/métodos , Proteômica/métodos , Interface Usuário-Computador , Internet , Metabolômica/normas , Proteômica/normas
7.
Anal Chem ; 91(5): 3302-3310, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30688441

RESUMO

Mass spectrometry (MS) is one of the primary techniques used for large-scale analysis of small molecules in metabolomics studies. To date, there has been little data format standardization in this field, as different software packages export results in different formats represented in XML or plain text, making data sharing, database deposition, and reanalysis highly challenging. Working within the consortia of the Metabolomics Standards Initiative, Proteomics Standards Initiative, and the Metabolomics Society, we have created mzTab-M to act as a common output format from analytical approaches using MS on small molecules. The format has been developed over several years, with input from a wide range of stakeholders. mzTab-M is a simple tab-separated text format, but importantly, the structure is highly standardized through the design of a detailed specification document, tightly coupled to validation software, and a mandatory controlled vocabulary of terms to populate it. The format is able to represent final quantification values from analyses, as well as the evidence trail in terms of features measured directly from MS (e.g., LC-MS, GC-MS, DIMS, etc.) and different types of approaches used to identify molecules. mzTab-M allows for ambiguity in the identification of molecules to be communicated clearly to readers of the files (both people and software). There are several implementations of the format available, and we anticipate widespread adoption in the field.


Assuntos
Metabolômica/métodos , Software , Bases de Dados Factuais , Espectrometria de Massas
8.
Anal Chem ; 89(22): 12252-12260, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29087685

RESUMO

Multiple-tracer approaches for investigating glucose metabolism in humans usually involve the administration of stable and radioactive glucose tracers and the subsequent determination of tracer enrichments in sampled blood. When using conventional, low-resolution mass spectrometry (LRMS), the number of spectral interferences rises rapidly with the number of stable tracers employed. Thus, in LRMS, both computational effort and statistical uncertainties associated with the correction for spectral interferences limit the number of stable tracers that can be simultaneously employed (usually two). Here we show that these limitations can be overcome by applying high-resolution mass spectrometry (HRMS). The HRMS method presented is based on the use of an Orbitrap mass spectrometer operated at a mass resolution of 100 000 to allow electrospray-generated ions of the deprotonated glucose molecules to be monitored at their exact masses. The tracer enrichment determination in blood plasma is demonstrated for several triple combinations of 13C- and 2H-labeled glucose tracers (e.g., [1-2H1]-, [6,6-2H2]-, [1,6-13C2]glucose). For each combination it is shown that ions arising from 2H-labeled tracers are completely differentiated from those arising from 13C-labeled tracers, thereby allowing the enrichment of a tracer to be simply calculated from the observed ion intensities using a standard curve with curve parameters unaffected by the presence of other tracers. For each tracer, the HRMS method exhibits low limits of detection and good repeatability in the tested 0.1-15.0% enrichment range. Additionally, due to short sample preparation and analysis times, the method is well-suited for high-throughput determination of multiple glucose tracer enrichments in plasma samples.


Assuntos
Deutério/química , Glucose/análise , Isótopos de Carbono , Glucose/metabolismo , Humanos , Espectrometria de Massas
9.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(8): 740-746, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28341148

RESUMO

Over the last two decades, lipidomics has evolved into an 'omics' technology pari passu with benchmarking 'omics' technologies, such as genomics or proteomics. The driving force behind this development was a constant advance in mass spectrometry and related technologies. The aim of this opinion article is to give the interested reader a concise but still comprehensive overview about the technological state of the art in lipidomics, current challenges and perspectives for future development. As such, this article guides through the whole workflow of lipidomics, from sampling to data analysis. This article is part of a Special Issue entitled: BBALIP_Lipidomics Opinion Articles edited by Sepp Kohlwein.


Assuntos
Metabolismo dos Lipídeos/fisiologia , Lipídeos/química , Genômica/métodos , Humanos , Espectrometria de Massas/métodos , Metabolômica/métodos , Proteômica/métodos , Manejo de Espécimes , Fluxo de Trabalho
10.
Mol Cell Proteomics ; 13(10): 2765-75, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24980485

RESUMO

The HUPO Proteomics Standards Initiative has developed several standardized data formats to facilitate data sharing in mass spectrometry (MS)-based proteomics. These allow researchers to report their complete results in a unified way. However, at present, there is no format to describe the final qualitative and quantitative results for proteomics and metabolomics experiments in a simple tabular format. Many downstream analysis use cases are only concerned with the final results of an experiment and require an easily accessible format, compatible with tools such as Microsoft Excel or R. We developed the mzTab file format for MS-based proteomics and metabolomics results to meet this need. mzTab is intended as a lightweight supplement to the existing standard XML-based file formats (mzML, mzIdentML, mzQuantML), providing a comprehensive summary, similar in concept to the supplemental material of a scientific publication. mzTab files can contain protein, peptide, and small molecule identifications together with experimental metadata and basic quantitative information. The format is not intended to store the complete experimental evidence but provides mechanisms to report results at different levels of detail. These range from a simple summary of the final results to a representation of the results including the experimental design. This format is ideally suited to make MS-based proteomics and metabolomics results available to a wider biological community outside the field of MS. Several software tools for proteomics and metabolomics have already adapted the format as an output format. The comprehensive mzTab specification document and extensive additional documentation can be found online.


Assuntos
Bases de Dados de Proteínas , Software , Acesso à Informação , Espectrometria de Massas , Metabolômica , Proteômica , Interface Usuário-Computador
11.
J Lipid Res ; 56(10): 1972-84, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26330055

RESUMO

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/ß-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.


Assuntos
Aciltransferases/metabolismo , Células Estreladas do Fígado/metabolismo , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Adipócitos/metabolismo , Animais , Linhagem Celular , Lipólise , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Perilipina-2 , Proteoma/metabolismo , Ratos , Proteínas de Ligação ao Retinol/metabolismo , Ésteres de Retinil , Triglicerídeos/metabolismo , Vitamina A/farmacologia
12.
Brief Bioinform ; 14(3): 375-90, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-22764120

RESUMO

Lipidomics, the systematic study of the lipid composition of a cell or tissue, is an invaluable complement to knowledge gained by genomics and proteomics research. Mass spectrometry provides a means to detect hundreds of lipids in parallel, and this includes low abundance species of lipids. Nevertheless, frequently occurring isobaric and isomeric lipid species complicate lipidomics analyses from an analytical and bioinformatics perspective. Various MS/MS strategies have evolved to resolve ambiguous identifications of lipid species, and these strategies have been supported by corresponding bioinformatics analysis tools. This review intends to familiarize readers with available bioinformatics MS/MS analysis tools and databases, the structural information obtainable from these, and their applicability to different MS/MS strategies. Finally, future challenges in detecting double bond positions are investigated from a bioinformatics perspective.


Assuntos
Biologia Computacional , Lipídeos/química , Ensaios de Triagem em Larga Escala , Estrutura Molecular , Espectrometria de Massas em Tandem
13.
Mol Cell Proteomics ; 12(1): 120-31, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23082028

RESUMO

Malaria morbidity and mortality caused by both Plasmodium falciparum and Plasmodium vivax extend well beyond the African continent, and although P. vivax causes between 80 and 300 million severe cases each year, vivax transmission remains poorly understood. Plasmodium parasites are transmitted by Anopheles mosquitoes, and the critical site of interaction between parasite and host is at the mosquito's luminal midgut brush border. Although the genome of the "model" African P. falciparum vector, Anopheles gambiae, has been sequenced, evolutionary divergence limits its utility as a reference across anophelines, especially non-sequenced P. vivax vectors such as Anopheles albimanus. Clearly, technologies and platforms that bridge this substantial scientific gap are required in order to provide public health scientists with key transcriptomic and proteomic information that could spur the development of novel interventions to combat this disease. To our knowledge, no approaches have been published that address this issue. To bolster our understanding of P. vivax-An. albimanus midgut interactions, we developed an integrated bioinformatic-hybrid RNA-Seq-LC-MS/MS approach involving An. albimanus transcriptome (15,764 contigs) and luminal midgut subproteome (9,445 proteins) assembly, which, when used with our custom Diptera protein database (685,078 sequences), facilitated a comparative proteomic analysis of the midgut brush borders of two important malaria vectors, An. gambiae and An. albimanus.


Assuntos
Anopheles/genética , Biologia Computacional , Proteínas de Insetos/análise , Insetos Vetores/genética , Proteoma/análise , RNA/análise , Sequência de Aminoácidos , Animais , Anopheles/parasitologia , Cromatografia Líquida , Bases de Dados de Proteínas , Interações Hospedeiro-Parasita , Humanos , Proteínas de Insetos/química , Insetos Vetores/parasitologia , Malária/parasitologia , Microvilosidades , Plasmodium falciparum , Plasmodium vivax , Proteômica , Espectrometria de Massas em Tandem , Transcriptoma
14.
Int J Mol Sci ; 16(4): 8351-63, 2015 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-25874761

RESUMO

A novel liquid chromatography-mass spectrometry (LC-MS) approach for analysis of oxidized phosphatidylcholines by an Orbitrap Fourier Transform mass spectrometer in positive electrospray ionization (ESI) coupled to hydrophilic interaction liquid chromatography (HILIC) was developed. This method depends on three selectivity criteria for separation and identification: retention time, exact mass at a resolution of 100,000 and collision induced dissociation (CID) fragment spectra in a linear ion trap. The process of chromatography development showed the best separation properties with a silica-based Kinetex column. This type of chromatography was able to separate all major lipid classes expected in mammalian samples, yielding increased sensitivity of oxidized phosphatidylcholines over reversed phase chromatography. Identification of molecular species was achieved by exact mass on intact molecular ions and CID tandem mass spectra containing characteristic fragments. Due to a lack of commercially available standards, method development was performed with copper induced oxidation products of palmitoyl-arachidonoyl-phosphatidylcholine, which resulted in a plethora of lipid species oxidized at the arachidonoyl moiety. Validation of the method was done with copper oxidized human low-density lipoprotein (LDL) prepared by ultracentrifugation. In these LDL samples we could identify 46 oxidized molecular phosphatidylcholine species out of 99 possible candidates.


Assuntos
Fosfatidilcolinas/química , Cromatografia Líquida de Alta Pressão , Análise de Fourier , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipoproteínas LDL/química , Oxirredução , Fosfatidilcolinas/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
15.
J Lipid Res ; 54(8): 2185-2194, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23740967

RESUMO

We showed earlier that nutritional stress like starvation or high-fat diet resulted in phenotypic changes in the lipidomes of hepatocyte lipid droplets (LDs), representative for the pathophysiological status of the mouse model. Here we extend our former study by adding genetic stress due to knockout (KO) of adipocyte triglyceride lipase (ATGL), the rate limiting enzyme in LD lipolysis. An intervention trial for 6 weeks with male wild-type (WT) and ATGL-KO mice was carried out; both genotypes were fed lab chow or were exposed to short-time starvation. Isolated LDs were analyzed by LC-MS/MS. Triacylglycerol, diacylglycerol, and phosphatidylcholine lipidomes, in that order, provided the best phenotypic signatures characteristic for respective stresses applied to the animals. This was evidenced at lipid species level by principal component analysis, calculation of average values for chain-lengths and numbers of double bonds, and by visualization in heat maps. Structural backgrounds for analyses and metabolic relationships were elaborated at lipid molecular species level. Relating our lipidomic data to nonalcoholic fatty liver diseases of nutritional and genetic etiologies with or without accompanying insulin resistance, phenotypic distinction in hepatocyte LDs dependent on insulin status emerged. Taken together, lipidomes of hepatocyte LDs are sensitive responders to nutritional and genetic stress.


Assuntos
Dano ao DNA , Hepatócitos/metabolismo , Lipase/deficiência , Lipídeos , Animais , Deleção de Genes , Hepatócitos/química , Lipase/genética , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tamanho da Partícula
16.
J Lipid Res ; 53(10): 2141-2152, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22872753

RESUMO

Liver steatosis can be induced by fasting or high-fat diet. We investigated by lipidomic analysis whether such metabolic states are reflected in the lipidome of hepatocyte lipid droplets (LDs) from mice fed normal chow diet (FED), fasted (FAS), or fed a high-fat diet (HFD). LC-MS/MS at levels of lipid species profiles and of lipid molecular species uncovered a FAS phenotype of LD enriched in triacylglycerol (TG) molecular species with very long-chain (VLC)-PUFA residues and an HFD phenotype with less unsaturated TG species in addition to characteristic lipid marker species. Nutritional stress did not result in dramatic structural alterations in diacylglycerol (DG) and phospholipid (PL) classes. Moreover, molecular species of bulk TG and of DG indicated concomitant de novo TG synthesis and lipase-catalyzed degradation to be active in LDs. DG species with VLC-PUFA residues would be preferred precursors for phosphatidylcholine (PC) species, the others for TG molecular species. In addition, molecular species of PL classes fitted the hepatocyte Kennedy and phosphatidylethanolamine methyltransferase pathways. We demonstrate that lipidomic analysis of LDs enables phenotyping of nutritional stress. TG species are best suited for such phenotyping, whereas structural analysis of TG, DG, and PL molecular species provides metabolic insights.


Assuntos
Hepatócitos/metabolismo , Lipídeos/análise , Fígado/metabolismo , Estresse Fisiológico , Animais , Dieta Hiperlipídica , Diglicerídeos/metabolismo , Jejum , Hepatócitos/química , Lipase/metabolismo , Fígado/química , Camundongos , Fosfatidilcolinas/metabolismo , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Triglicerídeos/metabolismo
17.
Bioinformatics ; 27(4): 572-7, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21169379

RESUMO

MOTIVATION: The accurate measurement of the lipidome permits insights into physiological and pathological processes. Of the present high-throughput technologies, LC-MS especially bears potential of monitoring quantitative changes in hundreds of lipids simultaneously. In order to extract valuable information from huge amount of mass spectrometry data, the aid of automated, reliable, highly sensitive and specific analysis algorithms is indispensable. RESULTS: We present here a novel approach for the quantitation of lipids in LC-MS data. The new algorithm obtains its analytical power by two major innovations: (i) a 3D algorithm that confines the peak borders in m/z and time direction and (ii) the use of the theoretical isotopic distribution of an analyte as selection/exclusion criterion. The algorithm is integrated in the Lipid Data Analyzer (LDA) application which additionally provides standardization, a statistics module for results analysis, a batch mode for unattended analysis of several runs and a 3D viewer for the manual verification. The statistics module offers sample grouping, tests between sample groups and export functionalities, where the results are visualized by heat maps and bar charts. The presented algorithm has been applied to data from a controlled experiment and to biological data, containing analytes distributed over an intensity range of 10(6). Our approach shows improved sensitivity and an extremely high positive predictive value compared with existing methods. Consequently, the novel algorithm, integrated in a user-friendly application, is a valuable improvement in the high-throughput analysis of the lipidome. IMPLEMENTATION AND AVAILABILITY: The Java application is freely available for non-commercial users at http://genome.tugraz.at/lda. Raw data associated with this manuscript may be downloaded from ProteomeCommons.org Tranche using the following hash: ZBh3nS5bXk6I/Vn32tB5Vh0qnMpVIW71HByFFQqM0RmdF4/4Hcn H3Wggh9kU2teYVOtM1JWwHIeMHqSS/bc2yYNFmyUAAAAAAACl DQ ==


Assuntos
Algoritmos , Cromatografia Líquida/métodos , Lipídeos/análise , Espectrometria de Massas/métodos , Animais , Hepatócitos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Software
18.
JACS Au ; 2(11): 2466-2480, 2022 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-36465531

RESUMO

Gangliosides are an indispensable glycolipid class concentrated on cell surfaces with a critical role in stem cell differentiation. Nonetheless, owing to the lack of suitable methods for scalable analysis covering the full scope of ganglioside molecular diversity, their mechanistic properties in signaling and differentiation remain undiscovered to a large extent. This work introduces a sensitive and comprehensive ganglioside assay based on liquid chromatography, high-resolution mass spectrometry, and multistage fragmentation. Complemented by an open-source data evaluation workflow, we provide automated in-depth lipid species-level and molecular species-level annotation based on decision rule sets for all major ganglioside classes. Compared to conventional state-of-the-art methods, the presented ganglioside assay offers (1) increased sensitivity, (2) superior structural elucidation, and (3) the possibility to detect novel ganglioside species. A major reason for the highly improved sensitivity is the optimized spectral readout based on the unique capability of two parallelizable mass analyzers for multistage fragmentation. We demonstrated the high-throughput universal capability of our novel analytical strategy by identifying 254 ganglioside species. As a proof of concept, 137 unique gangliosides were annotated in native and differentiated human mesenchymal stem cells including 78 potential cell-state-specific markers and 38 previously unreported gangliosides. A general increase of the ganglioside numbers upon differentiation was observed as well as cell-state-specific clustering based on the ganglioside species patterns. The combination of the developed glycolipidomics assay with the extended automated annotation tool enables comprehensive in-depth ganglioside characterization as shown on biological samples of interest. Our results suggest ganglioside patterns as a promising quality control tool for stem cells and their differentiation products. Additionally, we believe that our analytical workflow paves the way for probing glycolipid-based biochemical processes shedding light on the enigmatic processes of gangliosides and glycolipids in general.

19.
Food Chem ; 371: 131194, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34600364

RESUMO

Styrian pumpkin seed oil is a conditioned green-colored oil renowned for nutty smell and taste. Due to α-linolenic acid (ALA) contents below 1% of total fatty acids and the prospect of nutritional health claims based on its potential oxidation products, we investigated the fate of ALA and product oxylipins in the course of down-stream processing of seeds and in oils. Lipidomic analyses with Lipid Data Analyzer 2.8.1 revealed: Processing did not change (1) main fatty acid composition in the oils, (2) amounts of triacylglycerol species, (3) structures of triacylglycerol molecular species containing ALA. (4) Minor precursor ALA in fresh Styrian and normal pumpkins produced 6 product phytoprostanes in either cultivar, quantitatively more in the latter. (5) In oil samples 7 phytoprostanes and 2 phytofurans were detected. The latter two are specific for their presence in pumpkin seed oils, of note, quantitatively more in conditioned oils than in cold-pressed native oils.


Assuntos
Cucurbita , Ácidos Graxos , Lipidômica , Estrutura Molecular , Oxilipinas , Óleos de Plantas , Sementes , Triglicerídeos , Ácido alfa-Linolênico
20.
Metabolites ; 12(4)2022 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-35448474

RESUMO

This study centered on detecting potentially anti-inflammatory active constituents in ethanolic extracts of Chinese Lonicera species by taking an UHPLC-HRMS-based metabolite profiling approach. Extracts from eight different Lonicera species were subjected to both UHPLC-HRMS analysis and to pharmacological testing in three different cellular inflammation-related assays. Compounds exhibiting high correlations in orthogonal projections to latent structures discriminant analysis (OPLS-DA) of pharmacological and MS data served as potentially activity-related candidates. Of these candidates, 65 were tentatively or unambiguously annotated. 7-Hydroxy-5,3',4',5'-tetramethoxyflavone and three bioflavonoids, as well as three C32- and one C34-acetylated polyhydroxy fatty acid, were isolated from Lonicera hypoglauca leaves for the first time, and their structures were fully or partially elucidated. Of the potentially active candidate compounds, 15 were subsequently subjected to pharmacological testing. Their activities could be experimentally verified in part, emphasizing the relevance of Lonicera species as a source of anti-inflammatory active constituents. However, some compounds also impaired the cell viability. Overall, the approach was found useful to narrow down the number of potentially bioactive constituents in the complex extracts investigated. In the future, the application of more refined concepts, such as extract prefractionation combined with bio-chemometrics, may help to further enhance the reliability of candidate selection.

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