Transmission-Mode MALDI Mass Spectrometry Imaging of Single Cells: Optimizing Sample Preparation Protocols

Area of Science:

• Biophysics
• Analytical Chemistry
• Cell Biology

Background:

• Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) allows simultaneous visualization of numerous biomolecules in tissues and cells.
• Advancements in desorption/ionization techniques enable MALDI-MSI at cellular and subcellular resolutions.
• Effective sample preparation is critical for achieving high spatial resolution in MALDI-MSI.

Purpose of the Study:

• To investigate key sample preparation parameters for MALDI-MSI.
• To optimize protocols for preserving cellular morphology and molecular integrity.
• To evaluate the impact of washing, drying, fixation, and matrix coating on spatial resolution.

Main Methods:

• Optimization of chemical fixation (formalin incubation), washing, drying, and matrix coating steps.
• Utilized four model cell lines cultured on indium tin oxide (ITO)-coated glass slides.
• Employed transmission (t-)mode MALDI-2-MSI on a Q Exactive plus Orbitrap mass spectrometer at a 2 μm pixel size.
• Complementary analysis using phase contrast light microscopy and scanning electron microscopy.

Main Results:

• A protocol involving 5 min formalin incubation, isotonic washing, and mild drying demonstrated robust preservation of cell morphology and amine-containing phospholipids.
• Chemical fixation increased cell membrane permeability, leading to potential cytosolic compound leakage.
• Demonstrated the trade-offs between different sample preparation steps.

Conclusions:

• Developed and validated a robust sample preparation protocol for high-resolution MALDI-MSI.
• The protocol effectively preserves cellular morphology and key molecular species for single-cell analysis.
• This work contributes to advancing single-cell MALDI imaging for characterizing molecular heterogeneity.