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Volume determination with two standards allows absolute quantification and improved chemometric analysis of metabolites by NMR from submicroliter samples.

ABSTRACT: The accurate measurement of metabolite concentrations in miniscule biological sample volumes is often desirable, yet it remains challenging. In many cases, the starting analyte volumes are imprecisely known, or not directly measurable, and hence absolute metabolite concentrations are difficult to calculate. Here, we introduce volume determination using two standards (VDTS) as a general quantitative method for the analysis of polar metabolites in submicrolitre samples using (1)H NMR spectroscopy. This approach permits the back calculation of absolute metabolite concentrations from small biological samples of unknown volume. Where small sample volumes are also variable, VDTS can improve multivariate chemometric analysis. In this context, principal component analysis (PCA) yielded more logically consistent and biologically insightful outputs when we used volume-corrected spectra, calculated using VDTS, rather than probabilistic quotient normalization (PQN) of raw spectra. As proof-of-principle, the VDTS-based method and PCA were used to analyze polar metabolites in the hemolymph (blood) extracted from larvae of the very small but widely used genetic model organism Drosophila. This analysis showed that the hemolymph metabolomes of males and females are markedly different when larvae are well fed. However, gender-specific metabolomes tend to converge when larval dietary nutrients are restricted. We discuss the biological implications of these surprising results and compare and contrast them to previous analyses of Drosophila hemolymph and mammalian blood plasma. Together, these findings reveal an interesting and hitherto unknown sexual dimorphism in systemic Drosophila metabolites, clearly warranting further biological investigation. Importantly, the VDTS approach should be adaptable to many different analytical platforms, including mass spectrometry.

SUBMITTER: Ragan TJ

PROVIDER: S-EPMC3871890 | biostudies-other | 2013 Dec

REPOSITORIES: biostudies-other

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Volume determination with two standards allows absolute quantification and improved chemometric analysis of metabolites by NMR from submicroliter samples.

Ragan Timothy J TJ Bailey Andrew P AP Gould Alex P AP Driscoll Paul C PC

Analytical chemistry 20131122 24

The accurate measurement of metabolite concentrations in miniscule biological sample volumes is often desirable, yet it remains challenging. In many cases, the starting analyte volumes are imprecisely known, or not directly measurable, and hence absolute metabolite concentrations are difficult to calculate. Here, we introduce volume determination using two standards (VDTS) as a general quantitative method for the analysis of polar metabolites in submicrolitre samples using (1)H NMR spectroscopy. ...[more]

PMID: 24251761

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Project description:BackgroundQuantification of transcripts, proteins, or metabolites is straightforward when the factor used to normalize these values remains constant between samples. However, normalization factors often vary among samples and thus must be developed for each new analytical method.ResultsWe demonstrate quantification of transcript and protein levels in Arabidopsis based on genomic DNA copy number. We extracted total nucleic acid from 3-week-old rosette leaves of wild-type Arabidopsis and the pale-green/dwarf mutant, abc4, and quantified the number of transcripts by quantitative reverse-transcription PCR using genomic DNA copy number and ploidy (as determined by cytometry) for normalization. Our data indicated that normalization using genes commonly employed as references resulted in inaccuracies in transcript levels of the genes RBC-L and RBC-S (encoding the large and small subunits, respectively, of ribulose 1,5-bisphosphate carboxylase/oxygenase) in wild type and mutant. Normalization using genomic DNA copy number and ploidy, however, appropriately showed that the RBC-L and RBC-S transcript levels per cell in the mutant were significantly lower than that in wild type. Furthermore, quantification revealed that a cell of a 3-week-old wild-type Arabidopsis rosette leaf had an average of 7.5 × 103 transcripts of RBC-L, 9.9 × 103 transcripts of RBC-S, and 1.4 × 106 18S rRNA. We similarly analyzed the accumulation of RBC-L and LHCP (light-harvesting chlorophyll a/b protein) in wild type and mutant based on ploidy and genomic DNA copy number that was determined by direct quantitative PCR analysis of extracts using a DNA polymerase tolerant to a wide range of common PCR inhibitors. Furthermore, we estimated the number of RBC-L molecules (2.63 × 108) and chlorophyll molecules (1.85 × 109) in each cell in 3-week-old wild-type rosette leaves; these values had relatively low coefficients of variation, underscoring the reliability of our method.ConclusionGenomic DNA copy number and ploidy are useful as general normalization factors, providing an easy method for determining the number of transcripts, proteins, and metabolites in a cell.

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Volume determination with two standards allows absolute quantification and improved chemometric analysis of metabolites by NMR from submicroliter samples.

Publications

Volume determination with two standards allows absolute quantification and improved chemometric analysis of metabolites by NMR from submicroliter samples.

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