The extracellular matrix (ECM) provides structural scaffolding and mediates signaling in the extracellular space. According to our findings, approximately 75-85% of the fibrillar ECM resides in a chaotrope resistant insoluble ECM fraction (iECM), making proteomic characterization difficult without additional chemical digestion. We have developed optimized protocols for extraction of ECM that have been refined using samples from many systems, allowing for molecular-scale assessment of iECM proteome composition and architecture. We currently use these methods with the help of collaborators to assess ECM remodeling in pulmonary hypertension, anti-aging models, and tumor progression.
Image: David S. Goodsell, the Scripps Research Institute
CROSSLINKING MASS SPECTROMETRY
Cross-linking mass spectrometry (CL-MS) has quickly become a widely used method to map protein-protein interactions and provide distance constraints to elucidate topology in multi-protein complexes using chemical cross-linking reagents. CL-MS is a complimentary technique for NMR, cryo-EM, and X-ray crystallography based structural determination. With a combination of zero-length, MS-cleavable, and non-cleavable cross-linkers, we can provide structural information that cannot be determined via any other technique. In addition, CL-MS has proven useful for determining the structure of complexes too heterogenous for cryo-EM and too large for NMR.
FIBRIN CLOT STRUCTURE
The role of specific factor XIIIa crosslinks
on thrombus formation, regression, or probability for embolization are largely unknown. A molecular understanding of fibrin architecture at the level of these crosslinks could support the development of therapeutic strategies to prevent the sequelae of thromboembolism. We have developed a mass spectrometry method to map native factor XIIIa cross-links from the insoluble matrix component of whole blood or plasma fibrin clots and in vivo thrombi.
UNDERSTANDING BRAIN EVOLUTION
Olduvai domains have been identified as the largest human lineage specific increase in copy number of any protein coding sequence within the human genome. A strong correlation has been identified between increasing Olduvai copy number and increasing brain size and cortical neuron number within great apes, as well as macrocephaly and autism in humans. We are using a variety of biochemical techniques, including chemical cross-linking, BioID, and NMR, to probe the functional role of Olduvai domain-containing proteins within human brain development.
Image: Krienen et. al., Trends Cogn Sci, 2013
Using our ECM extraction methodology coupled with mass spectrometry, we are working to compose a comprehensive atlas describing tissue-specific ECM composition and architecture across different organs in three organisms (mouse, pig, and human). Due to recent technological advances, tissue engineering has become one of the fastest growing areas of medical science and technology in the US today. To design improved, organ-optimized ECM scaffolds, it is essential to develop a better model of how ECM components are distributed and organized across various organs. A comprehensive ECM Atlas will also provide a baseline for understanding ECM alterations during disease progression, wound healing, and aging. (Image: tissues.jensenlab.org)