Long-Term Metabolomics Reference Material

Abstract

The use of quality control samples in metabolomics ensures data quality, reproducibility, and comparability between studies, analytical platforms, and laboratories. Long-term, stable, and sustainable reference materials (RMs) are a critical component of the quality assurance/quality control (QA/QC) system; however, the limited selection of currently available matrix-matched RMs reduces their applicability for widespread use. To produce an RM in any context, for any matrix that is robust to changes over the course of time, we developed iterative batch averaging method (IBAT). To illustrate this method, we generated 11 independently grown Escherichia coli batches and made an RM over the course of 10 IBAT iterations. We measured the variance of these materials by nuclear magnetic resonance (NMR) and showed that IBAT produces a stable and sustainable RM over time. This E. coli RM was then used as a food source to produce a Caenorhabditis elegans RM for a metabolomics experiment. The metabolite extraction of this material, alongside 41 independently grown individual C. elegans samples of the same genotype, allowed us to estimate the proportion of sample variation in preanalytical steps. From the NMR data, we found that 40% of the metabolite variance is due to the metabolite extraction process and analysis and 60% is due to sample-to-sample variance. The availability of RMs in untargeted metabolomics is one of the predominant needs of the metabolomics community that reach beyond quality control practices. IBAT addresses this need by facilitating the production of biologically relevant RMs and increasing their widespread use.

Figure 1.

Iterative batch average method (IBAT). Batches of material are represented by columns (same-colored squares and letters). Rows represent homogeneous aliquots from each batch. Examples of sequential batch combinations are rows shaded from blue to purple. The right panel illustrates the IBAT-generated pools from individual batches. IBAT is only limited by the number of individual batches produced and can be adjusted to the number of aliquots required and to any material.

Figure 2.

(A) Untargeted full-resolution ¹H NMR profile of E. coli and spectral expansion between 6.8 and 7.2 ppm. NMR spectra in gray or orange correspond to IBAT or individual batches, respectively. (B) Radial plot representing the coefficient of variation (CV) for annotated metabolites using the same colors. The length of spokes corresponds to the CV of each metabolite. (C) Each data point represents the mean-centered peak height in each sample. Experimental IBAT samples are depicted in orange, and individual batches are depicted in gray. Cyan data points represent the simulated metabolite peak heights per number of averaged batches. Light gray-shaded areas represent ±1 standard deviation from the mean. Iva, isovalerate; Leu, leucine; Val, valine; Ile, isoleucine; 3 Hba, 3-hydroxybutyrate; Lac, lactate; Cad, cadaverine; AcOH, acetate; Glu, glutamate; Met, methionine; Asp, aspartate; Bet, betaine; Rib, ribose; Ura, uracil; Fum, fumarate; Tyr, tyrosine; Phe, phenylalanine; niacin, nicotinic acid; and Form, formate.

Figure 3.

Schematic overview of the C. elegans reference material production. The reference strain PD1074 nematodes were seeded from cryopreserved stocks and fed an E. coli RM (Supporting Methods). The harvested material from each bioreactor was washed, aliquoted, and stored. Aliquots from each reactor iteration were combined to produce a stable C. elegans reference material. This material can be divided into different sized aliquots according to the downstream application needs.

Figure 4.

Boxplots of pairwise standardized Euclidean distances. Each boxplot represents the distribution of distances from one sample to all of the other samples of the same group. Mean and median distances for each sample are indicated by markers. Blue-colored boxplots represent PD1074 samples that were processed in each block. The three pooled PD1074 samples were created from the samples in blocks 1 + 2, 3 + 4, and 5 + 6, respectively. C. elegans RM samples were generated using IBAT and processed alongside the PD1074 samples, one per block.