Extracellular Matrix Proteomics
The extracellular matrix (ECM) is the structural scaffolding for cells and a major signaling component of the microenvironment. The ECM is composed of a diverse set of proteins that cover a large range of molecular weights, form large aggregate structures, can be heavily glycosylated, crosslinked, and otherwise post-transnationally modified. Several of these properties contribute to insolubility and proteolytic resistance that make ECM proteins challenging to analyze using standard proteomic methods. We are developing optimized ECM methods and have analyzed a diverse range of samples using these methods:
Tumor Cell Metastasis
One key factor in determining whether cancer cell metastasis occurs is the state of the tumor microenvironment. Recent key studies have shown that the composition of the surrounding extracellular matrix (ECM) can determine metastatic outcome, specifically whether a tumor cell will transition from a non-invasive to an invasive phenotype. However, the specific molecular factors for generating these changes are poorly understood. We are developing proteomic methods, 3-dimensional cell culture model systems and functional assays to identify candidate ECM components and their modifications that influence cancer cell phenotype. Our goal is to validate top candidates in translational models and clinical samples for the identification of novel therapeutic interventions.
Mapping Transglutaminase Crosslinks
Tissue transglutaminase (TG2) generates protein-protein crosslinks (Q-K) in a wide range of proteins including those of the ECM. Due to the role of crosslinking in tissue development, homeostasis and cancer progression, determining the substrate sites of TG2 mediated crosslinking is a goal of our group. We are also interested in understanding the mechanisms by which TG2 targets these specific residues, and the factors that determine protein specificity. This requires identification of specific substrate sites and ultimately in vivo -generated crosslinked peptides.
Probing Protein Structure with MS
The goal of this project is to develop a robust method for mapping solvent accessible protein surfaces using chemical modification. By labeling a protein with and without ligand, antibody or other protein interaction partner we can perform a differential MS footprinting experiment. Side-chain protection can be used to identify conformational changes and interaction surfaces. This approach is similar to hydrogen deuterium exchange mass spectrometry (HXMS) but does not suffer from back exchange issues. We are currently working with a leading pharmaceutical company on protein interactions that have proven difficult to map with more traditional approaches.
Acute Hemorrhagic Shock and Trauma
Trauma remains the leading cause of years of life lost in the United States. Severe injuries involving hemorrhagic shock, fractures and other blunt or penetrating soft tissue destruction, can continue to threaten survival even after initial stabilization of a patient. We are working with the Colorado Trauma Research Group to:
- Identify novel mediators of multiple organ failure from mesenteric lymph (the conduit of toxic factors to target organs; lungs, liver, heart, etc.)
- Identify and interrogate toxic factors in blood products to decrease transfusion related mortalities
- Determine intracellular protein interactions involved in neutrophil priming events