Determining the role of stromal proteins in cancer biology has remained difficult due to a lack of tools. Current tools are qualitative and cannot report on protein sequence variation that is critical to understanding how stromal proteins help or impede cancer growth and metastasis. Our previous work has shown that bacterial collagenases can be used to produce stromal proteomes detected by mass spectrometry as 2D distributions on cancer tissues. We still lack tools that can focus down on target proteins for detailed studies of sequence variation and posttranslational modification. We propose using matrix metalloproteinases (MMPs) as enzymatic tools for targeted detection of stromal protein sequences in the cancer tissue microenvironment. Aim 1, we will develop and optimize a suite of MMPs for analysis of stromal sequences in tumor tissue sections. We will produce and optimize recombinant MMPs specifically for use on 2D solid substrates of tissue sections. We will integrate sequence information with current qualitative tools, expanding capabilities for stromal protein sequence detection down to a single cell niche. In Aim 2, we will focus on developing standards for quantitative detection of target stromal proteins from the tissue microenvironment. We will test the developed technology against a small but relevant cohort of emergent hepatocellular carcinoma from cirrhosis. Our research team has a unique combination of expertise for the production, development, and future commercialization of MMPs. The proposal is transformative since it will allow detection of stromal protein sequence variation from fixed clinical specimens which is impossible with conventional methods. A long-term objective is to significantly improve the community's ability to target stromal proteins within the tumor tissue microenvironment for use in basic research, directing therapies, or prognostic or diagnostic biomarkers.