In MALDI-Mass Spectrometry Imaging on Formalin-Fixed Paraffin-Embedded Tissue Specimen Section Thickness Significantly Influences m/z Peak Intensity

Abstract

Background: In matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) standardized sample preparation is important to obtain reliable results. Herein, the impact of section thickness in formalin-fixed paraffin embedded (FFPE) tissue microarrays (TMA) on spectral intensities is investigated.

Patients and methods: TMAs consisting of ten different tissues represented by duplicates of ten patients (n = 200 cores) are cut at 1, 3, and 5 μm. MSI analysis is performed and mean intensities of all evaluable cores are extracted. Measurements are merged and mean m/z intensities are compared.

Results: Visual inspection of spectral intensities between 1, 3, and 5 μm reveals generally higher intensities in thinner tissue sections. Specifically, higher intensities are observed in the vast majority of peaks (98.6%, p < 0.01) in 1 μm compared with 5 μm sections. Note that 28.4% and 2.1% of m/z values exhibit a at least two- and threefold intensity difference (p < 0.01) in 1 μm compared to 5 μm sections, respectively.

Conclusion: A section thickness of 1 μm results in higher spectral intensities compared with 5 μm. The results highlight the importance of standardized protocols in light of recent efforts to identify clinically relevant biomarkers using MSI. The use of TMAs for comparative analysis seems advantageous, as section thickness displays less variability.

Keywords: FFPE; MALDI-mass spectrometry imaging; proteomics; sample preparation; tissue section thickness.