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Novel protocol for spatial proteome analysis of formalin fixed paraffin embedded tissues

ABSTRACT: Novel In-insert FFPE proteomics combines single glass insert FFPE tissue processing with spatial quantitative data-independent mass spectrometry (DIA). In-insert proteomics represents a blueprint in spatial FFPE tissue subsection processing by completely omitting the robotic platform, desalting prior to injection to the liquid chromatograph and detergent removal while restraining protein losses to the minimum. In-insert proteomics has a sufficient sensitivity to preserve spatial context within a single FFPE tissue slide. Spatial pathway regulation trends were evaluated between 17 individual breast tissue voxels by the method. Bioinformatic spatial analysis of the most dysregulated proteins was performed to reveal hotspots of certain biochemical signaling/processes. A special attention was given to cytoskeleton reorganization as an effect of hormone signaling.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Breast

DISEASE(S): Breast Cancer

SUBMITTER: Jakub Faktor

LAB HEAD: Sachin Kote

PROVIDER: PXD037609 | Pride | 2024-04-19

REPOSITORIES: Pride

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Project description:Emiliania huxleyi: Cellular cascades induced by bacterial algicides Interactions between phytoplankton and bacteria play a central role in mediating oceanic biogeochemical cycling and microbial trophic structure in the ocean. The intricate relationships between these two domains of life are mediated via excreted molecules that facilitate communication and determine competitive outcomes. Yet, despite their predicted importance, identifying these secreted compounds and understanding their ecological significance has remained a challenge. Research in the Whalen Lab endeavors to (i) identify those bacterially-derived chemical signaling compounds (i.e. infochemicals) that mediate phytoplankton population dynamics, and (ii) determine the underlying physiological processes that contribute to phytoplankton tolerance or susceptibility to these compounds. Recently, the Whalen lab isolated an alkylquinolone-signaling molecule with known quorum sensing function from the globally distributed marine γ-proteobacteria, Pseudoalteromonas sp. capable of inducing species-specific phytoplankton mortality. This research was the first to suggest quorum sensing compounds have expanded and previously unrecognized ecological roles in regulating primary production and phytoplankton bloom dynamics. We are now investigating in how this alkylquinolone induces phytoplankton mortality via transcriptomic profiling and diagnostic biochemical analysis. Complementary to this transcriptomic examination, we will complete whole-cell proteomic approach to identify those phytoplankton proteins crucial in competitive interactions with bacterial infochemicals, but whose functions may not yet be known. With this proteomic approach in parallel to our transcriptomic investigation, we can establish a better understanding of the eukaryotic macromolecular targets and cellular cascades induced in response to bacterial algicides like alkylquinolones. With the knowledge gained from both approaches we can begin to address how these ?keystone molecules? influence population dynamics and community composition of phytoplankton and bacteria in field-based experiments with the goal of defining a new mechanistic framework for how bacterially derived signaling molecules influence biogeochemical cycles. D= DMSO - control treatment L= low 1 nm HHG additions M= medium 10 nm HHG additions H= high 100 nm HHG additions Each treatment had 4 biological replicates A-D

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Novel protocol for spatial proteome analysis of formalin fixed paraffin embedded tissues

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