Identification and High-Resolution Imaging of α-Tocopherol from Human Cells to Whole Animals by TOF-SIMS Tandem Mass Spectrometry

Abstract

A unique method for identification of biomolecular components in different biological specimens, while preserving the capability for high speed 2D and 3D molecular imaging, is employed to investigate cellular response to oxidative stress. The employed method enables observing the distribution of the antioxidant α-tocopherol and other molecules in cellular structures via time-of-flight secondary ion mass spectrometry (TOF-SIMS (MS¹)) imaging in parallel with tandem mass spectrometry (MS²) imaging, collected simultaneously. The described method is employed to examine a network formed by neuronal cells differentiated from human induced pluripotent stem cells (iPSCs), a model for investigating human neurons in vitro. The antioxidant α-tocopherol is identified in situ within different cellular layers utilizing a 3D TOF-SIMS tandem MS imaging analysis. As oxidative stress also plays an important role in mediating inflammation, the study was expanded to whole body tissue sections of M. marinum-infected zebrafish, a model organism for tuberculosis. The TOF-SIMS tandem MS imaging results reveal an increased presence of α-tocopherol in response to the pathogen. Graphical Abstract ᅟ.

Keywords: Mass spectrometry imaging; TOF-SIMS; Tandem MS; Vitamin E; α-tocopherol.

Graphical Abstract

ᅟ

Figure 1

Mass spectra collected by TOF-SIMS tandem MS imaging in the positive ion mode, plotted in the range of m/z 0–920. (a) An MS¹ precursor ion spectrum acquired from a cell culture of human neuronal cells prior to the 3D analysis. (b) The MS¹ precursor ion spectrum summed over the entire 3D analysis. The m/z 430.40 precursor peak is no longer present. The orange marker highlights the distorted portion of the spectrum surrounding the monoisotopic precursor selection window. (c) The MS² product ion spectrum, with precursor selection centered at m/z 430.40, summed over the entire 3D analysis. Some of the characteristic product ions, and the precursor, are annotated with their tentative structures. (d) An MS¹ precursor ion spectrum acquired from a thin tissue section of zebrafish infected with M. marinum bacteria. The m/z 430.20 precursor peak is no longer present, and the orange marker highlights the distorted portion of the spectrum surrounding the monoisotopic precursor selection window. (e) The MS² product ion spectrum, with precursor selection centered at m/z 430.20. Some of the characteristic product ions, and the precursor, are annotated with their tentative structures

Figure 2

TOF-SIMS tandem MS imaging of the human iPSC-derived neurons in the positive ion mode. The top row are MS¹ images and the bottom row are MS² images which were collected simultaneously during the in-depth voxel imaging analysis. The maximum counts per pixel are given in each panel. The ion images in the top row include the total ion count (TIC) (a), phosphocholine at m/z 184 (b), presumed phospholipid fragment ion and/or a diacyl glycerol ion with a total fatty acid composition of (32:0) at m/z 551 (c), and presumed [M+K]⁺ ion of PC(32:0) at m/z 772 (d) produced from the MS¹ data. The ion images in the bottom row include the TIC (all product ions and unfragmented precursor ions) of α-tocopherol (e), the prominent C₁₀H₁₃O₂⁺ product ion of α-tocopherol at m/z 165 (f), a minor product ion of α-tocopherol at m/z 57 (g), and a K⁺ product ion at m/z 39 (h). Note that the K⁺ ions are not localized to the cells only, but are more disbursed throughout the image area

Figure 3

3D TOF-SIMS tandem MS imaging of the human iPSC-derived neurons in the positive ion mode. Each 2D image in a row was extracted from the 3D image volume. The top row displays MS¹ images of the phosphocholine head group (C₅H₁₅NPO₄⁺), and the bottom row displays MS² TIC images of a molecule identified as the [M]⁺ of α-tocopherol, as a function of sputtered depth. The MS¹ and MS² data were collected simultaneously. The maximum counts per pixel are given in each panel. The soma and neurites of the cell are clearly observed in both the MS¹ and the MS² images

Figure 4

Histology of hematoxylin and eosin (H & E) stained sections from the abdomen of a healthy uninfected adult zebrafish (a) and a diseased adult zebrafish infected by M. marinum (b), showing severe granulomatous infection. The granulomas are visible in the H & E stain image as lighter structures with clearly visible cores which are indicated with small white arrows. TOF-SIMS (MS¹) total ion current (TIC) images, acquired in the positive ion polarity, of healthy (c) and diseased (d) zebrafish tissue sections. The sections used for MS imaging analysis were consecutive sections adjacent to those used for histology. The major organ groups are identified. TOF-SIMS (MS¹) images of healthy (e) and diseased (f) zebrafish tissue sections produced from positive polarity ions at m/z 430 which were identified by tandem MS (MS²) imaging as α-tocopherol. The green box in panel (f) indicates the approximate location of the tandem MS analysis for identification of the m/z 430 precursor ions