Profiling the tumor microenvironment proteome in prostate cancer using laser capture microdissection coupled to LC–MS—A technical report

• A systematic workflow for unbiased label-free LC–MS/MS analysis of laser capture microdissected material is presented.
• This LCM proteomics workflow was applied to fresh frozen tissue sections from prostate cancer patients.
• Expert annotation of regions of tumor for dissection from the sections of heterogenous tissue was undertaken by telepathology.
• Requirements for sample loading to support reproducible semi-quantitative analysis of protein expression were determined.
• To support tumor characterisation the LCM proteomics workflow was used to identify approximately 2000 proteins from tumor regions acquired from patient tissue sections.

Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections. Here, we present an optimized workflow for coupling LCM to LC–MS/MS including: sectioning of tissue, a standard LCM workflow, protein digestion and advanced LC–MS/MS. Soluble proteins extracted from benign epithelial cells, their associated stroma, tumor epithelial cells and their associated stromal cells from a single patient tissue sample were digested and profiled using advanced LC–MS/MS. The correlation between technical replicates was R2 = 0.99 with a mean % CV of 9.55% ± 8.73. The correlation between sample replicates was R2 = 0.97 with a mean % CV of 13.83% ± 10.17. This represents a robust, systematic approach for profiling of the tumor microenvironment using LCM coupled to label-free LC–MS/MS.

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