Laser capture microdissection and native mass spectrometry for spatially-resolved analysis of intact protein assemblies in tissue

Abstract

Previously, we have shown that native ambient mass spectrometry imaging allows the spatial mapping of folded proteins and their complexes in thin tissue sections. Subsequent top-down native ambient mass spectrometry of adjacent tissue section enables protein identification. The challenges associated with protein identification by this approach are (i) the low abundance of proteins in tissue and associated long data acquisition timescales and (ii) irregular spatial distributions which hamper targeted sampling of the relevant tissue location. Here, we demonstrate that these challenges may be overcome through integration of laser capture microdissection in the workflow. We show identification of intact protein assemblies in rat liver tissue and apply the approach to identification of proteins in the granular layer of rat cerebellum.

Fig. 1. (A) H&E stained section of rat liver tissue post-LCMD analysis. The enlarged region shows 6 separate sampling regions of interest that were obtained. Four larger regions (0.04 mm²) and two smaller regions (0.01 mm²) are shown cut from a 10 μm thick rat liver section. (B) H&E stained section of rat liver tissue showing the contact LESA sampled region (0.7 mm²).

Fig. 2. Full scan mass spectrum acquired over 2 minutes (resolution = 120k FWHM at m/z 200, standard pressure mode) of a 0.01 mm² region of interest captured from a 10 μm thick rat liver section.

Fig. 3. Full scan mass spectrum obtained following LCMD in the granular layer (A) and white matter (B) of the cerebellum of a 10 μm thick rat brain section. Laser captured regions from the granular layer (C) and white matter (D) are shown in the H&E images by the dotted black regions. Scale bars = 200 μm.