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INTRODUCTION: The metabolomics quality assurance and quality control consortium (mQACC) is enabling the identification, development, prioritization, and promotion of suitable reference materials (RMs) to be used in quality assurance (QA) and quality control (QC) for untargeted metabolomics research. OBJECTIVES: This review aims to highlight current RMs, and methodologies used within untargeted metabolomics and lipidomics communities to ensure standardization of results obtained from data analysis, interpretation and cross-study, and cross-laboratory comparisons. The essence of the aims is also applicable to other 'omics areas that generate high dimensional data. RESULTS: The potential for game-changing biochemical discoveries through mass spectrometry-based (MS) untargeted metabolomics and lipidomics are predicated on the evolution of more confident qualitative (and eventually quantitative) results from research laboratories. RMs are thus critical QC tools to be able to assure standardization, comparability, repeatability and reproducibility for untargeted data analysis, interpretation, to compare data within and across studies and across multiple laboratories. Standard operating procedures (SOPs) that promote, describe and exemplify the use of RMs will also improve QC for the metabolomics and lipidomics communities. CONCLUSIONS: The application of RMs described in this review may significantly improve data quality to support metabolomics and lipidomics research. The continued development and deployment of new RMs, together with interlaboratory studies and educational outreach and training, will further promote sound QA practices in the community.
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Lipidómica , Metabolómica , Espectrometría de Masas/métodos , Metabolómica/métodos , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
Modern biomarker and translational research as well as personalized health care studies rely heavily on powerful omics' technologies, including metabolomics and lipidomics. However, to translate metabolomics and lipidomics discoveries into a high-throughput clinical setting, standardization is of utmost importance. Here, we compared and benchmarked a quantitative lipidomics platform. The employed Lipidyzer platform is based on lipid class separation by means of differential mobility spectrometry with subsequent multiple reaction monitoring. Quantitation is achieved by the use of 54 deuterated internal standards and an automated informatics approach. We investigated the platform performance across nine laboratories using NIST SRM 1950-Metabolites in Frozen Human Plasma, and three NIST Candidate Reference Materials 8231-Frozen Human Plasma Suite for Metabolomics (high triglyceride, diabetic, and African-American plasma). In addition, we comparatively analyzed 59 plasma samples from individuals with familial hypercholesterolemia from a clinical cohort study. We provide evidence that the more practical methyl-tert-butyl ether extraction outperforms the classic Bligh and Dyer approach and compare our results with two previously published ring trials. In summary, we present standardized lipidomics protocols, allowing for the highly reproducible analysis of several hundred human plasma lipids, and present detailed molecular information for potentially disease relevant and ethnicity-related materials.
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Laboratorios , Lipidómica , Estudios de Cohortes , Humanos , Estándares de Referencia , Análisis EspectralRESUMEN
INTRODUCTION: The metabolomics quality assurance and quality control consortium (mQACC) evolved from the recognized need for a community-wide consensus on improving and systematizing quality assurance (QA) and quality control (QC) practices for untargeted metabolomics. OBJECTIVES: In this work, we sought to identify and share the common and divergent QA and QC practices amongst mQACC members and collaborators who use liquid chromatography-mass spectrometry (LC-MS) in untargeted metabolomics. METHODS: All authors voluntarily participated in this collaborative research project by providing the details of and insights into the QA and QC practices used in their laboratories. This sharing was enabled via a six-page questionnaire composed of over 120 questions and comment fields which was developed as part of this work and has proved the basis for ongoing mQACC outreach. RESULTS: For QA, many laboratories reported documenting maintenance, calibration and tuning (82%); having established data storage and archival processes (71%); depositing data in public repositories (55%); having standard operating procedures (SOPs) in place for all laboratory processes (68%) and training staff on laboratory processes (55%). For QC, universal practices included using system suitability procedures (100%) and using a robust system of identification (Metabolomics Standards Initiative level 1 identification standards) for at least some of the detected compounds. Most laboratories used QC samples (>86%); used internal standards (91%); used a designated analytical acquisition template with randomized experimental samples (91%); and manually reviewed peak integration following data acquisition (86%). A minority of laboratories included technical replicates of experimental samples in their workflows (36%). CONCLUSIONS: Although the 23 contributors were researchers with diverse and international backgrounds from academia, industry and government, they are not necessarily representative of the worldwide pool of practitioners due to the recruitment method for participants and its voluntary nature. However, both questionnaire and the findings presented here have already informed and led other data gathering efforts by mQACC at conferences and other outreach activities and will continue to evolve in order to guide discussions for recommendations of best practices within the community and to establish internationally agreed upon reporting standards. We very much welcome further feedback from readers of this article.
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Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Metabolómica/métodos , Humanos , Laboratorios , Control de Calidad , Proyectos de Investigación , Encuestas y CuestionariosRESUMEN
The diagnosis and treatment of diseases such as cancer is becoming more accurate and specialized with the advent of precision medicine techniques, research and treatments. Reaching down to the cellular and even sub-cellular level, diagnostic tests can pinpoint specific, individual information from each patient, and guide providers to a more accurate plan of treatment. With this advanced knowledge, researchers and providers can better gauge the effectiveness of drugs, radiation, and other therapies, which is bound to lead to a more accurate, if not more positive, prognosis. As precision medicine becomes more established, new techniques, equipment, materials and testing methods will be required. Herein, we will examine the recent innovations in assays, devices and software, along with next generation sequencing in genomics diagnostics which are in use or are being developed for personalized medicine. So as to avoid duplication and produce the fullest possible benefit, all involved must be strongly encouraged to collaborate, across national borders, public and private sectors, science, medicine and academia alike. In this paper we will offer recommendations for tools, research and development, along with ideas for implementation. We plan to begin with discussion of the lessons learned to date, and the current research on pharmacogenomics. Given the steady stream of advances in imaging mass spectrometry and nanoLC-MS/MS, and use of genomic, proteomic and metabolomics biomarkers to distinguish healthy tissue from diseased cells, there is great potential to utilize pharmacogenomics to tailor a drug or drugs to a particular cohort of patients. Such efforts very well may bring increased hope for small groups of non-responders and those who have demonstrated adverse reactions to current treatments.
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Genómica/métodos , Neoplasias/terapia , Medicina de Precisión/métodos , Humanos , PronósticoRESUMEN
We describe here the agreed upon first development steps and priority objectives of a community engagement effort to address current challenges in quality assurance (QA) and quality control (QC) in untargeted metabolomic studies. This has included (1) a QA and QC questionnaire responded to by the metabolomics community in 2015 which recommended education of the metabolomics community, development of appropriate standard reference materials and providing incentives for laboratories to apply QA and QC; (2) a 2-day 'Think Tank on Quality Assurance and Quality Control for Untargeted Metabolomic Studies' held at the National Cancer Institute's Shady Grove Campus and (3) establishment of the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) to drive forward developments in a coordinated manner.
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Metabolómica/métodos , Metabolómica/normas , Humanos , Laboratorios , Control de Calidad , Mejoramiento de la CalidadRESUMEN
Lipidomics - the global assessment of lipids - can be performed using a variety of mass spectrometry (MS)-based approaches. However, choosing the optimal approach in terms of lipid coverage, robustness and throughput can be a challenging task. Here, we compare a novel targeted quantitative lipidomics platform known as the Lipidyzer to a conventional untargeted liquid chromatography (LC)-MS approach. We find that both platforms are efficient in profiling more than 300 lipids across 11 lipid classes in mouse plasma with precision and accuracy below 20% for most lipids. While the untargeted and targeted platforms detect similar numbers of lipids, the former identifies a broader range of lipid classes and can unambiguously identify all three fatty acids in triacylglycerols (TAG). Quantitative measurements from both approaches exhibit a median correlation coefficient (r) of 0.99 using a dilution series of deuterated internal standards and 0.71 using endogenous plasma lipids in the context of aging. Application of both platforms to plasma from aging mouse reveals similar changes in total lipid levels across all major lipid classes and in specific lipid species. Interestingly, TAG is the lipid class that exhibits the most changes with age, suggesting that TAG metabolism is particularly sensitive to the aging process in mice. Collectively, our data show that the Lipidyzer platform provides comprehensive profiling of the most prevalent lipids in plasma in a simple and automated manner.
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Envejecimiento/sangre , Envejecimiento/metabolismo , Lípidos/sangre , Plasma/metabolismo , Animales , Cromatografía Liquida/métodos , Estudios de Evaluación como Asunto , Masculino , Ratones , Ratones Endogámicos C57BL , Espectrometría de Masas en Tándem/métodos , Triglicéridos/sangreRESUMEN
Lipids play a key role in the signaling pathways of cancer, cardiovascular, diabetic, and inflammatory diseases. A major challenge in the analysis of lipids is the many isobaric interferences present in highly complex samples that confound identification and accurate quantitation. After obtaining the total lipid extract from a sample, differential mobility separation has proven to be a powerful tool for gas-phase fractionation of lipid classes. When combined with mass spectrometry, this allows the unambiguous identification and thus quantification of lipid molecular species. These components, sample extraction, gas-phase separation, and mass spectrometry, form the basis of a novel integrated quantitative lipid analysis platform.
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Lípidos/sangre , Espectrometría de Masas en Tándem/métodos , Fraccionamiento Químico , Humanos , Transducción de SeñalRESUMEN
An autonomous metabolomic workflow combining mass spectrometry analysis with tandem mass spectrometry data acquisition was designed to allow for simultaneous data processing and metabolite characterization. Although previously tandem mass spectrometry data have been generated on the fly, the experiments described herein combine this technology with the bioinformatic resources of XCMS and METLIN. As a result of this unique integration, we can analyze large profiling datasets and simultaneously obtain structural identifications. Validation of the workflow on bacterial samples allowed the profiling on the order of a thousand metabolite features with simultaneous tandem mass spectra data acquisition. The tandem mass spectrometry data acquisition enabled automatic search and matching against the METLIN tandem mass spectrometry database, shortening the current workflow from days to hours. Overall, the autonomous approach to untargeted metabolomics provides an efficient means of metabolomic profiling, and will ultimately allow the more rapid integration of comparative analyses, metabolite identification, and data analysis at a systems biology level.
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Biología Computacional , Desulfovibrio vulgaris/metabolismo , Procesamiento Automatizado de Datos/métodos , Metabolómica/métodos , Cromatografía Liquida/métodos , Bases de Datos Factuales , Desulfovibrio vulgaris/crecimiento & desarrollo , Programas Informáticos , Espectrometría de Masas en Tándem/métodosRESUMEN
The analysis of lipids by mass spectrometry (MS) can provide in-depth characterization for many forms of biological samples. However, such workflows can also be hampered by challenges like low chromatographic resolution for lipid separations and the convolution of mass spectra from isomeric and isobaric species. To address these issues, we describe the use of differential mobility spectrometry (DMS) as a rapid and predictable separation technique within a shotgun lipidomics workflow, with a special focus on phospholipids (PLs). These analytes, ionized by electrospray ionization (ESI), are filtered using DMS prior to MS analysis. The observed separation (measured in terms of DMS compensation voltage) is affected by several factors, including the m/z of the lipid ion, the structure of an individual ion, and the presence of chemical modifiers in the DMS cell. Such DMS separations can simplify the analysis of complex extracts in a robust and reproducible manner, independent of utilized MS instrumentation. The predictable separation achieved with DMS can facilitate correct lipid assignments among many isobaric and isomeric species independent of the resolution settings of the MS analysis. This leads to highly comprehensive and quantitative lipidomic outputs through rapid profiling analyses, such as Q1 and MRM scans. The ultimate benefit of the DMS separation in this unique shotgun lipidomics workflow is its ability to separate many isobaric and isomeric lipids that by standard shotgun lipidomics workflows are difficult to assess precisely, for example, ether and diacyl species and phosphatidylcholine (PC) and sphingomyelin (SM) lipids.
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Metabolismo de los Lípidos , Espectrometría de Masas/métodosRESUMEN
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed which, with sample preparation using a commercially available kit, allows rapid quantitation of 39 chloroformate-derivatised amino acids (AAs), polyamines (PAs) and dipeptides (DPs) in complex biological matrices. Lower limits of quantitation (LOQ) were 20-150nM for putrescine, spermine, spermidine, cadaverine, agmatine, and below 5µM for all analytes. Responses were linear for all analytes between 0.5 and 50µM. Quantitative measurements of all 39 metabolites were achieved within a 15min runtime. The method was evaluated with a Pseudomonas aeruginosa cell extract study (n=24) and a larger human urine study (n=308). Batch effects were observed in the urine study and an investigation of instrument and sample stability showed a wave-like pattern in the MS responses. Both the run order and inter-batch variation were successfully corrected by normalising to pooled urine quality control data. Thus, this method should be suitable for diverse biological matrices and for large as well as small sample sets.
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Aminoácidos/química , Cromatografía Liquida/métodos , Dipéptidos/química , Poliaminas/química , Pseudomonas aeruginosa/química , Espectrometría de Masas en Tándem/métodos , Aminoácidos/orina , Dipéptidos/orina , Formiatos/química , Humanos , Poliaminas/orina , Pseudomonas aeruginosa/metabolismoRESUMEN
BACKGROUND: COPD, a leading cause of mortality is currently assessed by spirometry (forced expiratory volume in 1 second, FEV(1)). However FEV(1) does not correlate with patient mortality. ECLIPSE (Evaluation of Chronic obstructive pulmonary disease to Longitudinally Identify Predictive Surrogate Endpoints) aims to identify biomarkers that correlate with clinically relevant COPD subtypes, and to assess how these may predict disease progression. New methods were developed and validated to evaluate small molecules as potential diagnostic tools in patients with COPD, COPD related cachexia and cancer related cachexia. METHODS AND FINDINGS: quantitative LC-MS/MS was developed to measure 34 amino acids and dipeptides for stratification of patient groups. Subsets of the ECLIPSE patients were used to assess biomarkers of lung obstruction; GOLD IV (n = 30) versus control (n = 30); emphysema (n = 38) versus airways disease (n = 21) and cachexia (n = 30) versus normal body mass index (n = 30). Serum from cachexic (n = 7) and non-cachexic (n = 5) pancreatic cancer patients were included as controls. Targeted LC-MS/MS distinguished GOLD IV patients from controls, patients with and without emphysema and patients with and without cachexia. Glutamine, aspartate and arginine were significantly increased (p < 0.05; FDR adjustment α < 0.1) in cachexia, emphysema and GOLD IV patients and aminoadipate was decreased. Several amino acid concentrations were significantly altered in patients with COPD but not patients with pancreatic cancer (serine, sarcosine, tryptophan, BCAAs and 3-methylhistdine). Increased γ-aminobutyrate (GABA, p < 0.01) levels were specific to cachexia in patients with pancreatic cancer. ß-aminoisobutyrate, 1-methylhistidine and asparagine (p < 0.05) were common across the patients with cachexia from both the COPD and pancreatic cancer cohorts. CONCLUSION: these results demonstrate that a metabolomic fingerprint has potential to stratify patients for the treatment of COPD and may provide a means of assessing response to therapy. GOLD IV patients were distinguished from smoker control subjects, patients with emphysema were distinguished from those without emphysema and COPD patients displaying cachexia were distinguished from those not displaying cachexia. General markers of cachexia were discovered reflecting both COPD- and pancreatic cancer-related cachexia (increased glutamine, aspartate, arginine, and asparagine and decreased aminoadipate, ß-aminoisobutyrate and 1-methylhistidine). Metabolomic biomarkers, particularly altered levels of GABA, could be exploited as a way of monitoring treatment efficacy and tumour recurrence for patients with pancreatic cancer experiencing cachexia.
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Aminoácidos/sangre , Caquexia/sangre , Metabolómica , Neoplasias Pancreáticas/sangre , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfisema Pulmonar/sangre , Anciano , Aminoácidos/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Índice de Masa Corporal , Caquexia/diagnóstico , Progresión de la Enfermedad , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/mortalidad , Enfisema Pulmonar/diagnóstico , Pruebas de Función Respiratoria , Índice de Severidad de la Enfermedad , Fumar , Espectrometría de Masas en Tándem , Ácido gamma-Aminobutírico/sangreRESUMEN
There is a paucity of biomarkers for chronic obstructive pulmonary disease (COPD). Metabolomics were applied to a defined COPD patient cohort from the ECLIPSE study (Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points). Results were correlated with accepted biomarkers for the disease. Baseline control serum (n=66) and Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II (n=70), III (n=64) and IV (n=44) COPD patients were analysed by proton nuclear magnetic resonance ((1)H NMR). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to confirm amino acid changes detected by (1)H NMR. Data were correlated with body composition, emphysema and systemic inflammation. (1)H NMR identified decreased lipoproteins, N,N-dimethylglycine, and increased glutamine, phenylalanine, 3-methylhistidine and ketone bodies in COPD patients with decreased branched-chain amino acids (BCAAs) observed in GOLD stage IV patients. BCAAs, their degradation products, 3-methylhistidine, ketone bodies, and triglycerides were correlated negatively with cachexia and positively with systemic inflammation. Emphysema patients also displayed decreased serum creatine, glycine and N,N-dimethylglycine. LC-MS/MS confirmed (1)H NMR findings relating to BCAAs, glutamine and 3-methylhistidine in GOLD stage IV patients. NMR-based metabolomics characterised COPD patients based on systemic effects and lung function parameters. Increased protein turnover occurred in all COPD patients with increased protein degradation in individuals with emphysema and cachexia.