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. 2022 Sep 20;94(37):12875-12883.
doi: 10.1021/acs.analchem.2c02918. Epub 2022 Sep 7.

A Direct Infusion Probe for Rapid Metabolomics of Low-Volume Samples

Cátia Marques  1 , Liangwen Liu  2 , Kyle D Duncan  1 , Ingela Lanekoff  1
Affiliations

A Direct Infusion Probe for Rapid Metabolomics of Low-Volume Samples

Cátia Marques et al. Anal Chem. .

Abstract

Targeted and nontargeted metabolomics has the potential to evaluate and detect global metabolite changes in biological systems. Direct infusion mass spectrometric analysis enables detection of all ionizable small molecules, thus simultaneously providing information on both metabolites and lipids in chemically complex samples. However, to unravel the heterogeneity of the metabolic status of cells in culture and tissue a low number of cells per sample should be analyzed with high sensitivity, which requires low sample volumes. Here, we present the design and characterization of the direct infusion probe, DIP. The DIP is simple to build and position directly in front of a mass spectrometer for rapid metabolomics of chemically complex biological samples using pneumatically assisted electrospray ionization at 1 μL/min flow rate. The resulting data is acquired in a square wave profile with minimal carryover between samples that enhances throughput and enables several minutes of uniform MS signal from 5 μL sample volumes. The DIP was applied to study the intracellular metabolism of insulin secreting INS-1 cells and the results show that exposure to 20 mM glucose for 15 min significantly alters the abundance of several small metabolites, amino acids, and lipids.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Design of the direct infusion probe (DIP). (A) Schematic overview of the probe in front of the mass spectrometer inlet and (B) photograph of the assembled probe with a 200 μL centrifuge tube.
Figure 2
Figure 2
Carryover and comparison between established sources. (A) Extracted ion chronograms of glutamate-15N (m/z 149.0575) in standard solution (blue) and glutamate (m/z 148.0604) in rat brain extract (red) with a rolling average of 5. (B and C) Fold change of relative LOD for selected metabolite standards between m/z 75 and 230 plotted for DIP compared to (B) ESI and (C) HESI. Positive values indicated better LOD with the DIP. Protonated adducts are black and sodiated adducts orange.
Figure 3
Figure 3
Single scan, without any averaged microscans, high resolution spectrum in positive ion mode from a methanolic rat brain extract analyzed with DIP-MS. (A) Full spectrum from 70 to 1000 Da, (B) same spectrum zoomed into m/z 100–206 and with several metabolites annotated, and (C) same spectrum zoomed into m/z 700–900 with several annotated lipids. A list of additional putatively annotated peaks is provided in Table S9.
Figure 4
Figure 4
Quantification of endogenous metabolites and lipids in samples with low cell densities. Concentrations of endogenous (A) small metabolites and (B) phospholipids from 5 μL samples with 20 to 500 cells/μL. Note that the Y-axes are for species depicted as squares (left) and circles (right). Error bars represent one standard deviation of triplicates of sample preparation. n = 3 per cell density, which equals 21 samples in total.
Figure 5
Figure 5
Nontargeted and quantitative targeted metabolite profiling of INS-1 cells exposed to either low (1 mM) or high (20 mM) glucose. (A) Nontargeted data analysis: Volcano plot originated from the global median normalization of a total of 190 features putatively assigned. A total of 56 metabolites were considered significant (p < 0.05) according to Student’s t test analysis and fold change higher than 1.04. Red and blue correspond to up and down regulated features, respectively, and in gray are the nonsignificant features. (B) Network: The global metabolomic network highlighting in black dots some of the significantly altered metabolites. (C) Targeted data analysis: Concentration of metabolites in INS-1 cell exposed to either low (light blue) or high (dark blue) glucose. All results are significantly different (p < 0.05) between the treatment groups according to two-tailed unpaired homoscedastic Student’s t test analysis. All results refer to the sodiated adducts of each metabolite, except for valine and proline where the protonated adducts are used. Error bars represent one standard deviation of n = 10 of sample preparation from the same batch.
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