PROTEOMICS – TECHNOLOGY DEVELOPMENT AND APPLICATIONS
We apply our expertise in proteomics by improving sample preparation methods for increasing sequence coverage and by developing new ways to profile proteins in complex mixtures.
Virtual 2D-Gel Electrophoresis/MS - The virtual two-dimensional gel electrophoresis/mass spectrometry (virtual 2D gel/MS) technology combines the premier, high-resolution capabilities of 2D gel electrophoresis with the sensitivity and high mass accuracy of mass spectrometry. Gel-wide chemical and enzymatic methods with further interrogation by MALDI-MS/MS provide identifications, sequence-related information, and post-translational/transcriptional modification information. The MS imaging-based virtual 2D gel/MS platform may potentially link the benefits of “top-down” and “bottom-up” proteomics.
Discovering New Protein Targets of Ligand Binding – With Professor Jing Huang (UCLA Pharmacology), we are developing a high-throughput LC-MS platform coupled to a universally applicable target identification approach to analyze direct small-molecule binding to its protein target(s). DARTS (drug affinity responsive target stability) relies on a well-known phenomenon in which ligand binding causes thermodynamic stabilization of its target protein’s structure such that the protein becomes resistant to a variety of insults, including proteolysis. DARTS allows the protein target of a ligand to be revealed without requiring modification or immobilization of the small molecule. Coupled with our improved MS platform, DARTS is a powerful method for discovering new protein targets and regulatory functions of drugs and metabolites.
1. Ogorzalek Loo RR, Cavalcoli JD, VanBogelen RA, Mitchell C, Loo JA, Moldover B, and Andrews PC. “Virtual 2-D Gel Electrophoresis: Visualization and Analysis of the E. coli Proteome by Mass Spectrometry.” Anal Chem 2001; 73: 4063-4070.
2. Ogorzalek Loo RR, Lam Y, Loo JA, and Schumaker VN. “Virtual 2-D Gel Electrophoresis of High Density Lipoproteins.” Electrophoresis 2004; 25: 2384-2391. 3. Erde J, Ogorzalek Loo RR, and Loo JA. “Enhanced FASP (eFASP) to Increase Proteome Coverage and Sample Recovery for Quantitative Proteomic Experiments.” J Proteome Res 2014; 13: 1885−1895.
4. Pai MY, Lomenick B, Hwang H, Schiestl R, McBride W, Loo JA, and Huang J. “Drug Affinity Responsive Target Stability (DARTS) for Small-Molecule Target Identification.” Methods Mol Biol 2015; 1263: 287-298.
5. Lohnes K, Quebbemann NR, Liu K, Kobzeff F, Loo JA, and Ogorzalek Loo RR. “Combining High-throughput MALDI-TOF Mass Spectrometry and Isoelectric Focusing Gel Electrophoresis for Virtual 2D Gel-based Proteomics.” Methods, in press.
HUMAN HEALTH-RELATED PROTEOMICS
Our lab has been intimately involved in several large-scale proteomics projects that relate to improved understanding of human health and diseases. Biofluids, such as saliva and bronchoalveolar and nasal lavage fluids, have been examined as a means to discover disease biomarkers. Tissue samples are used to probe the biological effects of ionizing radiation and to aid the development of radiomitigative drugs.
Traumatic Brain Injury - We use quantitative proteomics strategies to address the currently unmet need for protein markers of traumatic brain injury (TBI). TBI is the leading cause of mortality and morbidity in individuals under the age of 45 years. Current neurotrauma marker candidates are of limited clinical use because they poorly correlate with outcome. With Dr. Ina Wanner (UCLA Semel Institute), we are developing astrocyte-specific biomarkers that correlate to trauma severity. Our work potentially benefits neurotrauma research by delivering novel insight to brain cell injury mechanisms and tracking of their unique biofluid signatures.