組織内の代謝物の局在を調べる方法：組織切片＋質量分析

質量分析は試料中の物質を同定する方法ですが、生体の組織や細胞のどこにその物質があるかということに関しては、高分解能の情報は得られないものと思っていました。しかし切片から直接部分的にビームを当てて、イオン化して試料分析に逐次かけていくという技術が開発されていたようです。

Mass Spectrometry for Metabolomics Chemical & Engineering News チャンネル登録者数 2.72万人

Gooogle AIによる概要

In beam mass spectrometry for biological tissue section localization, a focused beam of energy or particles is directed at a tissue sample to desorb and ionize molecules. A mass spectrometer then analyzes these ions, with the location of each analysis point recorded to reconstruct a “molecular map” showing the spatial distribution of hundreds of different compounds in the tissue. This technique is broadly known as mass spectrometry imaging (MSI). 

Key beam-based MSI techniques for tissue analysis include:

Laser-based techniques

Matrix-assisted laser desorption/ionization (MALDI) MSI

  • Method: A thin tissue section is first coated with a crystalline matrix compound. A pulsed laser beam is then rastered across the surface, with the matrix absorbing the laser energy, which co-desorbs and ionizes the tissue’s molecules.
  • Resolution: Spatial resolution is determined by the size of the laser spot, typically 10 to 100 micrometers. Recent advancements using laser beam scanning have achieved resolutions as fine as 10 μm.
  • Applications: Used to map the distribution of a wide range of biomolecules, including lipids, peptides, and proteins. MALDI is often combined with high-resolution mass analyzers, such as Fourier transform ion cyclotron resonance (FT-ICR) MS, for exceptional mass accuracy. 

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

  • Method: A laser beam ablates—or vaporizes—a minute amount of tissue, which is then transported to an inductively coupled plasma (ICP). The plasma’s high temperature atomizes and ionizes the material, and a mass spectrometer quantifies the elemental isotopes present.
  • Resolution: Capable of producing high-resolution elemental maps, down to the micrometer or sub-micrometer level.
  • Applications: Used for imaging metals, metalloids, and other trace elements within tissues. It is particularly valuable for toxicology, and for studying metal-related diseases like neurodegenerative disorders. 

Ion beam-based techniques

Secondary ion mass spectrometry (SIMS)

  • Method: A focused beam of high-energy ions (the primary beam) bombards the tissue surface, causing secondary ions to be ejected. These secondary ions are collected and analyzed by a mass spectrometer.
  • Resolution: Known for its exceptional spatial resolution, with specialized NanoSIMS instruments achieving sub-100 nm resolution.
  • Applications: Used for high-resolution imaging of small molecules, such as lipids and metabolites, and for determining isotopic ratios. Its high vacuum requirement and surface sensitivity make it well-suited for single-cell analysis. 

Ambient ionization beam techniques

Desorption electrospray ionization (DESI)

  • Method: A charged spray of solvent microdroplets is directed at the tissue at ambient pressure. As the droplets hit the surface, they extract and ionize molecules, which are then analyzed by a mass spectrometer.
  • Resolution: Generally provides lower spatial resolution (typically 50–200 μm) compared to high-vacuum methods, but offers the advantage of minimal sample preparation.
  • Applications: Useful for rapid, real-time molecular profiling during surgical procedures (mass spectrometry-guided surgery) and for mapping drugs and metabolites. 

Nanospray desorption electrospray ionization (nano-DESI)

  • Method: Uses a liquid microjunction formed by two capillaries to continuously extract and analyze analytes from the tissue surface.
  • Resolution: An ambient technique that achieves high spatial resolution, with some reports demonstrating resolutions better than 10 μm.
  • Applications: Offers a high-sensitivity method for imaging proteoforms and other biomolecules, with the ability to perform high-resolution protein mapping. 

Workflow and applications

The general workflow for beam mass spectrometry on tissue sections involves several steps:

  1. Sample preparation: Freezing fresh tissue in liquid nitrogen followed by cutting thin sections (typically 10–20 μm) with a cryostat.
  2. Beam scanning: The tissue is placed on a conductive plate or slide and a beam is rastered across its surface.
  3. Spectral acquisition: A mass spectrum is generated for each pixel (location) analyzed by the beam.
  4. Data processing: Software converts the raw spectral data into visual, color-coded molecular maps showing the spatial distribution of specific molecules.
  5. Data integration: The resulting molecular images can be overlaid with traditional stained histology images to correlate molecular data with tissue pathology and microstructures. 

These techniques are widely used in biomedical research to identify biomarkers, investigate disease pathology (e.g., tumor margins), and study the biodistribution of drugs and metabolites.