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

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.

Global and Spatial Metabolomics of Individual Cells Using a Tapered Pneumatically Assisted nano-DESI Probe

“…We and others have previously reported metabolome alterations of INS-1 cells treated with low and high glucose prior to homogenization and bulk analysis. − Specifically, with bulk analysis we showed a large number of metabolites that were significantly altered between the two conditions using the direct infusion probe . Here, we investigate if metabolome alterations can also be detected in individual INS-1 cells under the same treatment conditions.…”

“…39−42 Specifically, with bulk analysis we showed a large number of metabolites that were significantly altered between the two conditions using the direct infusion probe. 43 Here, we investigate if metabolome alterations can also be detected in individual INS-1 cells under the same treatment conditions. Using the established optimized conditions, the tapered PA nano-DESI probe was used in touch-down mode to analyze 93 and 97 INS-1 cells treated with 1 or 20 mM glucose for 15 min, respectively.…”

Screening autism-associated environmental factors in differentiating human neural progenitors with fractional factorial design-based transcriptomics

“…Metabolomic profiling was performed using the direct infusion probe (DIP) for ESI-MS (Marques et al, 2022) on snap-frozen cell lysates (N=24) at day 5 of exposure to 3.0 μM FH during undirected differentiation of NES cells, with 3 technical replicates each for the treated and untreated groups in all four cell lines: CTRL Male , CTRL Female , ASD HNRNPU and ASD CASK . The cells were stored as pellets in -80 freezer prior to lysing with the electrospray solvent containing 9:1 methanol:water and 0.1% formic acid.…”

Design of metabolomic studies: pre-analytical process

“…Метаболомика -это комплексный анализ малых молекул, участвующих в метаболических путях, которые управляют биохимическими процессами и функциями клеток организма [1]. Метаболом наиболее полно отражает состояние организма человека под влиянием внутренних и внешних факторов, включая изменение окружающей среды, образ жизни, наличие или отсутствие заболеваний, прием лекарств, диету.…”

“…Основные преимущества масс-спектрометрии -высокая чувствительность и селективность. Метаболомные исследования проводят как методом масс-спектрометрии прямого ввода (direct infusion mass spectrometry) [1,6], так и с использованием гибридных методов с предварительным хроматографическим разделением -хроматомасс-спектрометрии [3,7,8]. граммное обеспечение, например, Metaboanalyst 5.0 [9,10].…”