SiMeEx, a simplified method for metabolite extraction of adherent mammalian cells

Abstract

A reliable method for metabolite extraction is central to mass spectrometry-based metabolomics. However, existing methods are lengthy, mostly due to the step of scraping cells from cell culture vessels, which restricts metabolomics in broader application such as lower cell numbers and high-throughput studies. Here, we present a simplified metabolite extraction (SiMeEx) method, to efficiently and quickly extract metabolites from adherent mammalian cells. Our method excludes the cell scraping step and therefore allows for a more efficient extraction of polar metabolites in less than 30 min per 12-well plate. We demonstrate that SiMeEx achieves the same metabolite recovery as using a standard method containing a scraping step, in various immortalized and primary cells. Omitting cell scraping does not compromise the performance of non-targeted and targeted GC-MS analysis, but enables metabolome analysis of cell culture on smaller well sizes down to 96-well plates. Therefore, SiMeEx demonstrates advantages not only on time and resources, but also on the applicability in high-throughput studies.

Keywords: GC-MS; mammalian cells; metabolite extraction; metabolomics; stable isotope labeling.

FIGURE 1

‘Standard’ vs SiMeEx. Schematic diagram of the workflows for the ‘standard’ (left) and SiMeEx (right) metabolite extraction.

FIGURE 2

SiMeEx is sufficient to stop metabolic activities and disrupt cellular and mitochondrial membranes. (A). Density plot of the metabolite extraction efficiency based on non-targeted GC-MS measurement. The metabolite extraction efficiency is defined as the log2-fold change of SiMeEx and the ‘standard’ method for the mean signals of each detected metabolite and biological replicate. (B). Comparison of relative standard errors during the extraction with SiMeEx and the ‘standard’ method. Means and SEM are shown for each cell line, calculated based on the non-targeted measurement. (C). Live/dead staining with calcein-AM (green, for esterase activity) and ethidium homodimer-1 (red, for DNA binding) of RAW 264.7 cells treated with 0.9% NaCl (Ctrl), ddH₂O, MeOH or MeOH + ddH₂O. (D). Abundance of isotopologues of central carbon metabolites in disrupted RAW 264.7 cells incubated with [U-¹³C₃]-sodium pyruvate. Data is normalized to isotopologue abundance from unlabeled pyruvate control samples. (E). Abundance of isotopologues of mitochondrial metabolites in RAW 264.7 cells incubated with [U-¹³C₆]-glucose, followed by ‘standard’ or SiMeEx extraction. (B,D,E), data are presented as mean ± SEM pooled from three independent experiments with n = 6–12 (RAW264.7 cells) or 3 (all other cells) technical replicates each.

FIGURE 3

Co-extraction of nucleic acids and proteins. (A,B). Distribution of recovered RNA (A) and protein (B) between interphase of extraction fluids (Interphase) and cellular remnants after extraction (Remnants), using the ‘standard’ and SiMeEx methods, and the data present the percentage distribution of Interphase and Remnants. (C,D). Yield of RNA (C) and protein (D) recovered from interphase of extraction fluids, using ‘standard’ or SiMeEx methods. All data are presented as mean ± SEM calculated from three independent experiments with n = 3 technical replicates. Significances were calculated by unpaired student’s t-test with p < 0.001: ***.

FIGURE 4

High-throughput applicability of SiMeEx. (A). Schematic workflow for SiMeEx extraction for 48- or 96-well plate formats. (B). Levels of representative metabolites extracted from RAW 264.7 cells cultured on different formats of multi-well plates. All data are normalized to metabolite levels of 12-well plate and presented as mean ± SEM calculated from three independent experiments with n = 3 technical replicates.