Quantitative mass spectrometry imaging of glutathione in healthy and cancerous hen ovarian tissue sections by infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI)

Abstract

A quantitative mass spectrometry imaging (QMSI) method for absolute quantification of glutathione (GSH) in healthy and cancerous hen ovarian tissues using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is presented. Using this technique, the ion abundance of GSH was normalized to that of a structural analogue, which was sprayed on the slide prior to mounting the tissue sections. This normalization strategy significantly improved the voxel-to-voxel variability; the variability is attributed to the overall ionization process. Subsequently, a series of calibration spots of stable isotope-labeled (SIL) GSH were pipetted on top of the tissue to construct a spatial calibration curve, and calculate the concentration of GSH in both tissue sections. The QMSI results were verified by LC-MS/MS quantification of GSH for the same tissues. GSH was extracted from tissue sections in a slightly acidic buffer and was then alkylated using N-ethylmaleimide to minimize autoxidation of GSH to glutathione disulfide. The alkylated GSH was separated from other contaminants using reversed phase liquid chromatography (RPLC) coupled to a triple quadrupole mass spectrometer, and the z-ion transition of NEM-GSH was used to quantify GSH in each tissue section. While the absolute values obtained using IR-MALDESI QMSI and LC-MS/MS were different, a ∼2-fold increase in the concentration of GSH in cancer tissue compared to the healthy tissue was observed using both techniques. Possible reasons for the difference between absolute concentration values obtained using IR-MALDESI QMSI and LC-MS/MS are also discussed.

The Space Dimension at the Micro Level: Mass Spectrometry Imaging of Drugs in Tissues

“…Like in LAESI, the plume of desorbed material interacts with an electrospray, where analytes are ionized (Sampson, Hawkridge, & Muddiman, 2006). The choice of the matrix is not as critical as in with MALDI since ice can be used universally for different tissue types and analytes without the drawback of matrix‐related peaks in the lower m / z region of the spectra (Robichaud et al, 2013; Nazari et al, 2018). IR‐MALDESI also offers optimal spatial resolution up to 50 µm (Bokhart et al, 2017)…”

Laser Desorption/Ablation Postionization Mass Spectrometry: Recent Progress in Bioanalytical Applications

“…A tutorial review describing the use of IR‐MALDESI for untargeted metabolic MSI analyses has been published (Bokhart & Muddiman, 2016). Recent reports on metabolic profiling using IR‐MALDESI include the following: optimization of the instrument parameters for mapping lipids, cholesterol, and small metabolites in mouse brain sections with improved ion abundances (Robichaud et al, 2014); identification of triterpenoid lipids and study of their spatial distribution in fermented cucumber with high salt concentration (Ekelöf et al, 2017); discriminating healthy and cancerous hen ovarian tissue from distribution and relative abundance of metabolites via polarity switching MSI (Nazari & Muddiman, 2016b) and from principal component analysis and multivariate curve resolution—alternating least squares resolved MSI data (Akbari Lakeh et al, 2019); again quantitative MSI of glutathione in healthy and cancerous hen ovarian, which revealed a ~2‐fold increase of glutathione concentration in the disease state (Nazari et al, 2018a); profiling underivatized neurotransmitters in rat brain tissues exposed to tetrabromobisphenol A, with MS images showing gender‐specific neurotransmitter distribution and level differences (Bagley et al, 2018); analysis of terpenes with optimized electrospray solvent composition and injection times (Nazari et al, 2018b). These reports demonstrate the feasibility of IR‐MALDESI to high throughput identification/quantification of biomolecules in complex matrixes.…”

“…For tissue and body fluid samples, the difficulty arises from interferences of abundant endogenous compounds and organic matrixes. However, emerging cases have brought success to quantitative imaging by the use of standards of known concentrations for linear regression analysis (Bokhart et al, 2015; Nazari et al, 2018a). For cases where suitable standards are not available, the development of reactive spray‐based LD/API techniques would be a potential solution.…”

Three-dimensional (3D) imaging of lipids in skin tissues with infrared matrix-assisted laser desorption electrospray ionization (MALDESI) mass spectrometry

“…Depth resolution was readily adjustable by altering the laser energy level, which allowed removal of sufficient material (depth resolution on the order of microns), suggesting that IR-MALDESI MSI would be a practical tool for 3D tissue examination in terms of the volume to be analyzed in a reasonable amount of time. Over the past decade, IR-MALDSI MSI has evolved as an innovative technique for mapping molecules ranging from lipids, proteins, metabolites to exogenous molecules with minimal preparation, high salt tolerance, and deeper coverage in a wide array of biological specimens including animal tissue sections [20][21][22][23][24][25]. To fully exploit the 3D potential of IR-MALDESI MSI, the current study applies IR-MALDESI MSI in 3D tissue imaging using skin, a multi-layer modality, as a proof of principle.…”