research

PROTEOMICS

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.

Representative publications

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.

Representative publications

1. Yan W, Apweiler R, Balgley BM, Boontheung P, Bundy JL, Cargile BJ, Cole S, Fang X, Gonzalez-Begne M, Griffin TJ, Hagen F, Hu S, Lee CS, Malamud D, Melvin JE, Menon R, Mueller M, Rhodus NL, Sevinsky JR, States D, Stephenson JL Jr, Than S, Yates III JR, Yu W, Xie H, Xie Y, Omenn GS, Loo JA, Wong DT. “Systematic Comparison of the Human Saliva and Plasma Proteomes.” Proteomics Clin Appl 2009; 3: 116-134.

2. Zhang L, Wang M, Kang X, Boontheung P, Li N, Nel AE, and Loo JA. “Oxidative Stress and Asthma: Proteome Analysis of Chitinase-like Proteins and FIZZ1 in Lung Tissue and Bronchoalveolar Lavage Fluid.” J Proteome Res 2009; 9: 1631-1638.

3. Kang X, Li N, Wang M, Boontheung P, Harkema JR, Bramble LA, Nel AE, and Loo JA. “Adjuvant Effects of Ambient Particulate Matter Monitored by Proteomics of Bronchoalveolar Lavage Fluid.” Proteomics 2010; 10: 520-531.

4. Loo JA, Yan W, Ramachandran P, and Wong DT. “Comparative Human Salivary and Plasma Proteomes.” J Dental Res 2010; 89: 1016-1023.

5. Shirasaki DI, Greiner ER, Al-Ramahi I, Gray M, Boontheung P, Geschwind DH, Botas J, Coppola G, Horvath S, Loo JA, and Yang XW. “Network Organization of the Huntingtin Proteomic Interactome in Mammalian Brain.” Neuron, 2012; 75: 41-57.

6. Hsieh SI, Castruita M, Malasarn D, Urzica E, Erde J, Page MD, Yamasaki H, Casero D, Pellegrini M, Merchant SS, and Loo JA. “The Proteome of Copper, Iron, Zinc, and Manganese Micronutrient Deficiency in Chlamydomonas reinhardtii.” Mol Cell Proteomics 2013; 12: 65-86.

7. Shen S, Ogorzalek Loo RR, Wanner I-B, and Loo JA. “Addressing the Needs of Traumatic Brain Injury with Clinical Proteomics.” Clin Proteomics 2014; 11: 11 (13 pp.).

8. Ferguson CN, Fowler JWM, Waxer JF, Gatti RA, and Loo JA. “Mass Spectrometry-Based Tissue Imaging of Small Molecules.” In Advancements of Mass Spectrometry in Biomedical Research, Woods AG and Darie CC, Eds., Springer: Switzerland, 2014, pp 283-299.