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Removal of optimal cutting temperature (O.C.T.) compound from embedded tissue for MALDI imaging of lipids
Jacob X M Truong1,2,3, Xander Spotbeen1,4, Jake White1,2
1South Australian Health and Medical Research Institute (SAHMRI), North Terrace, Adelaide, South Australia, 5000, Australia.
Abstract:
Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a common molecular imaging modality used to characterise the abundance and spatial distribution of lipids in situ. There are several technical challenges predominantly involving sample pre-treatment and preparation which have complicated the analysis of clinical tissues by MALDI-MSI. Firstly, the common embedding of samples in optimal cutting temperature (O.C.T.), which contains high concentrations of polyethylene glycol (PEG) polymers, causes analyte signal suppression during mass spectrometry (MS) by competing for available ions during ionisation. This suppressive effect has constrained the application of MALDI-MSI for the molecular mapping of clinical tissues. Secondly, the complexity of the mass spectra is obtained by the formation of multiple adduct ions. The process of analyte ion formation during MALDI can generate multiple m/z peaks from a single lipid species due to the presence of alkali salts in tissues, resulting in the suppression of protonated adduct formation and the generation of multiple near isobaric ions which produce overlapping spatial distributions. Presented is a method to simultaneously remove O.C.T. and endogenous salts. This approach was applied to lipid imaging in order to prevent analyte suppression, simplify data interpretation, and improve sensitivity by promoting lipid protonation and reducing the formation of alkali adducts.
Insights
This study presents a new method to improve lipid analysis in clinical tissues using matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI). The technique overcomes challenges from sample preparation, enhancing lipid detection and spatial distribution mapping.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Molecular Imaging
Background:
- Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is crucial for analyzing lipid distribution in tissues.
- Clinical tissue analysis using MALDI-MSI faces challenges due to sample embedding media like Optimal Cutting Temperature (O.C.T.) and endogenous salts.
- O.C.T. (containing polyethylene glycol) and salts cause signal suppression and complex mass spectra, hindering accurate lipid mapping.
Purpose of the Study:
- To develop a method for simultaneous removal of O.C.T. and endogenous salts from clinical tissue samples.
- To improve the sensitivity, data interpretation, and spatial resolution of lipid analysis in MALDI-MSI.
- To overcome common technical limitations in applying MALDI-MSI to clinical tissue samples.
Main Methods:
- A novel sample preparation technique was developed to eliminate O.C.T. embedding media and endogenous salts.
- The method was applied to lipid imaging of clinical tissue samples.
- Mass spectrometry imaging was used to analyze lipid abundance and spatial distribution.
Main Results:
- The new method effectively removed O.C.T. and endogenous salts, preventing analyte signal suppression.
- Simplified mass spectra were obtained by reducing the formation of multiple adduct ions and isobaric ions.
- Improved lipid protonation and reduced alkali adduct formation led to enhanced sensitivity and clearer spatial distributions.
Conclusions:
- The developed method significantly enhances the application of MALDI-MSI for clinical tissue lipid analysis.
- Simultaneous removal of O.C.T. and salts is key to overcoming major technical hurdles in the modality.
- This approach facilitates more accurate and sensitive molecular mapping of lipids in clinical samples.
