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Characterization of rapid extraction protocols for high-throughput metabolomics.

ABSTRACT: In the last five years, high-throughput metabolomics has significantly advanced scientific research and holds the potential to promote strides in the fields of clinical metabolomics and personalized medicine. While innovations in the field of flow-injection mass spectrometry and three-minute metabolomics methods now allow investigators to process hundreds to thousands of samples per day, time-sensitive clinical applications, particularly in the emergency department, are limited by a lack of rapid extraction methods.Here we characterized the efficacy of fast liquid-liquid extractions for characterization of hydrophilic compounds through ultra-high-pressure liquid chromatography/mass spectrometry. Internal stable-isotope-labeled standards were used to quantitatively characterize markers of energy and oxidative metabolism in human whole blood, plasma and red blood cells - three common matrices of clinical relevance.For all the tested matrices, vortexing time (4-60 min) did not significantly affect extraction yields for the tested hydrophilic metabolites. Coefficients of variations <<20% for all tested compounds, except for the redox-sensitive metabolite cystine (accumulating over time). Internal standards and second extractions confirmed recoveries >80% for all tested metabolites, except for basic amino acids and polyamines, which showed reproducible yields ranging from 50 to 75%. Global profiling and absolute quantitation of 24 metabolites revealed similarities between the plasma and red blood cell metabolomes.Rapid extraction (~4 min) of hydrophilic compounds is a viable and potentially automatable strategy to perform quantitative analysis of whole blood, plasma and red blood cells for research or clinical applications.

SUBMITTER: Gehrke S

PROVIDER: S-EPMC5547002 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Characterization of rapid extraction protocols for high-throughput metabolomics.

Gehrke Sarah S Reisz Julie A JA Nemkov Travis T Hansen Kirk C KC D'Alessandro Angelo A

Rapid communications in mass spectrometry : RCM 20170901 17

<h4>Rationale</h4>In the last five years, high-throughput metabolomics has significantly advanced scientific research and holds the potential to promote strides in the fields of clinical metabolomics and personalized medicine. While innovations in the field of flow-injection mass spectrometry and three-minute metabolomics methods now allow investigators to process hundreds to thousands of samples per day, time-sensitive clinical applications, particularly in the emergency department, are limited ...[more]

PMID: 28586533

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Project description:INTRODUCTION:When analyzing the human plasma metabolome with Nuclear Magnetic Resonance (NMR) spectroscopy, the Carr-Purcell-Meiboom-Gill (CPMG) experiment is commonly employed for large studies. However, this process can lead to compromised statistical analyses due to residual macromolecule signals. In addition, the utilization of Trimethylsilylpropanoic acid (TSP) as an internal standard often leads to quantification issues, and binning, as a spectral summarization step, can result in features not clearly assignable to metabolites. OBJECTIVES:Our aim was to establish a new complete protocol for large plasma cohorts collected with the purpose of describing the comparative metabolic profile of groups of samples. METHODS:We compared the conventional CPMG approach to a novel procedure that involves diffusion NMR, using the Longitudinal Eddy-Current Delay (LED) experiment, maleic acid (MA) as the quantification reference and peak picking for spectral reduction. This comparison was carried out using the ultrafiltration method as a gold standard in a simple sample classification experiment, with Partial Least Squares-Discriminant Analysis (PLS-DA) and the resulting metabolic signatures for multivariate data analysis. In addition, the quantification capabilities of the method were evaluated. RESULTS:We found that the LED method applied was able to detect more metabolites than CPMG and suppress macromolecule signals more efficiently. The complete protocol was able to yield PLS-DA models with enhanced classification accuracy as well as a more reliable set of important features than the conventional CPMG approach. Assessment of the quantitative capabilities of the method resulted in good linearity, recovery and agreement with an established amino acid assay for the majority of the metabolites tested. Regarding repeatability,?~?85% of all peaks had an adequately low coefficient of variation (<?30%) in replicate samples. CONCLUSION:Overall, our comparison yielded a high-throughput untargeted plasma NMR protocol for optimized data acquisition and processing that is expected to be a valuable contribution in the field of metabolic biomarker discovery.

Project description:BackgroundIn order to control and eradicate transboundary animal diseases, early diagnosis and reaction is essential for the implementation of control activities. Thus, mobile diagnostic units which allow analytical testing close to the site of occurrence could provide valuable support for centralized laboratories. Consequently, the availability of diagnostic tests using mobile amplification and detection technologies has been increasing over the past years. However, methods enabling rapid and simple nucleic acid extraction also under resource-limited settings are still scarce.MethodsIn the present study rapid extraction protocols based on magnetic particle technology have been developed. For this purpose, the two open extraction platforms KingFisher™ Duo (Thermo Fisher Scientific) and BioSprint® 15 (Qiagen) as well as the fully automated EZ1® advanced XL instrument (Qiagen) were used. All protocols were validated in comparison to standard manual extraction using blood and serum samples from animals infected with Schmallenberg virus or bovine viral diarrhea virus.ResultsAll newly developed protocols allowed a complete extraction within 30 minutes of time. The fully automated EZ1-extraction yielded the highest reproducibility, whereas slightly higher intra- and inter-assay variations were observed using the open platforms. Compared to the manual procedure, the analytical sensitivity of all the rapid protocols was 1 log10 step reduced for extraction from blood samples. For sera a reduced dynamic range could only be observed using the maximally shortened BioSprint 15 protocol. Validation using clinical samples showed an excellent concordance of all the rapid extraction protocols to the standard manual extraction procedure, independent of sample materials and target viruses.ConclusionsThe results of this study show that the speed-optimized novel extraction protocols allow rapid and simple nucleic acid extractions for a variety of target viruses without significant loss of sensitivity compared to standard procedures. For this reason they represent valuable tools to accelerate magnetic particle based automated extraction.

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Characterization of rapid extraction protocols for high-throughput metabolomics.

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Characterization of rapid extraction protocols for high-throughput metabolomics.

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