The goal of this research is to sample biomolecules from formalin fixed paraffin embedded (FFPE) tissue for subsequent qualitative and quantitative analyses using mass spectrometry (MS) based proteomics strategies. A thin tissue section is fixed to a microscope slide that is mounted on a support. A mid-infrared laser is focused to the back of the microscope slide (i.e., transmission mode) and an area of the tissue is ablated. The continuous laser beam is fixed in place and, using motorized translational stages, the microscope slide is moved along two axes. The ablated material is captured either by holding a collection tube below the slide or using a filter connected to rough vacuum. A camera equipped with a telescopic objective allows sample visualization. An in-house developed LabVIEW software program is used to control the laser, camera, and stages for ablation of selectively shaped regions of interest (ROI). Other imaging sources (e.g., microscope images of stained sections) are used for reference and co-registration purposes. Initial experiments were conducted with 8 µm thick rat liver tissue sections, which were ablated after the removal of paraffin and at a rate of 5 mm/s. Areas of ~25 mm2 were captured and the protein fraction was processed for LC-MS/MS shotgun proteomics analysis, which allowed identification of over 170 proteins. Current efforts are aimed at developing protocols to capture DNA and RNA for genomic sequencing and epigenetic analysis. While the proteomic experiments were conducted after a paraffin removal step, the possibility of capturing samples from sections embedded in the wax medium will also be explored. In addition, enzymatic activity of catalase (one of the enzymes detected in our experiments, which has been shown to be active when captured from fresh frozen tissue sections) will be evaluated.

Laser ablation sampling of formalin fixed paraffin embedded tissue

255th National Meeting (2018)

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