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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