Proteomic Technology for the Life Sciences
Our research is focused on the development and application of novel proteomic technology to facilitate biomedical research. Research projects involve mass spectrometry of proteins, peptides and other biomolecules that are relevant to biomedical questions, in particular to cancer-related diseases. We collaborate closely with other researchers around the world and within City of Hope in a truly interdisciplinary setting. Most our technology is available through the City of Hope Mass Spectrometry & Proteomics Core.
Emerging Infectious Diseases
| Figure 1. (click to enlarge)
Electron Micrograph of an
acquired at the City of
Hope Electron Microscopy
Patients who receive transplants of hematopoietic cells or solid organs or are treated for autoimmune diseases usually receive immunosuppressive medication. Such treatment is necessary to control graft-versus-host disease, graft rejection or autoinflammatory episodes, respectively.
Unfortunately, immunosuppression also increases the risk for opportunistic infections with all classes on infectious agents, and, as a result of more advanced and aggressive modern healthcare practices, the number of immunosuppressed patients is on the rise. One of the most severe emerging infectious diseases is invasive pulmonary aspergillosis, which is a rapidly progressing and most often fatal fungus infection that commonly occurs in hematopoietic stem cell transplant recipients.
Our research employs novel mass spectrometric technology to study the proteomes of fungal and bacterial pathogens with the goals being to develop effective vaccines and to detect infections at early stages when the response to medication and other interventions is more effective.
Discovery And Testing Of Anti-Fungal Vaccine Candidates
Pulmonary exposure to viable Aspergillus fumigatus spores protects mice (a CF-1 strain) against invasive aspergillosis when challenged under corticosteroid immunosuppression (Ito, JI and Lyons, JM, 2002, J. Infect. Dis.). Using a proteomic approach, we found that such immunized animals produce specific antibodies against the A. fumigatus protein Asp f 3, and most importantly, mice vaccinated with recombinant forms of Asp f 3 are protected. Asp f 3 is a known allergen with a described bi-partite IgE-binding epitope. The epitope can be disrupted without diminishing the protective effect of the resulting, modified recombinant Asp f 3, making it an interesting vaccine candidate for potential human use.
Sensitive detection of fungal and bacterial proteins of diagnostic potentia
The detection of low-level peptides by single-stage mass spectrometry is often impaired by a limited dynamic range and by suppression effects caused by so-called “chemical noise.” Hypothesis-driven multistage mass spectrometry (HMS-MS) allows us to overcome these limitations. The detection principle is based on a search for the presence or absence of a predicted peptide in a manner analogous to the use of an antigen-specific antibody. A novel matrix assisted laser desorption / ionization (MALDI) ion trap mass spectrometer is tuned on pre-calculated masses in order to collect and fragment ions that may hypothetically be present in a particular sample. Characteristic fragmentation patterns differentiate the signal from the background noise and provide reliable evidence for the presence of the predicted peptide. The detection of hypothesized proteolytic cleavage products can reveal the presence of putative proteins in a given sample. Such a targeted approach is useful for proteomic studies, e.g., when interacting proteins need to be found within a limited pool of potential binding partners. Additionally, the method can be used to search for the presence of certain post-translational modifications such as phosphorylation. We are applying the HMS-MS methods to detect the release of fungal proteins in patient specimens for potential diagnostic use.
Figure 2. (click to enlarge)
A) Reversed phase High Performance Liquid Chromatography (RP HPLC) of culture filtrate (CF) and hyphal sonicate (HS) and dot-blot analysis with sera of immunized mice. (B) MALDI-mass spectrometric analysis of a 19 kDa band of this fraction identifies A. fumigatus Asp f 3 as the major immunogenic protein component; black triangles mark assigned peptide ions that match the Asp f 3 sequence. Stars mark two peptide ions from a minor impurity, cofilin, that were assigned based on MS/MS data of these ions.
Detection of Microorganisms and Biodefense
Our laboratory is pursuing proteomic and molecular biological studies to improve the detection of pathogenic microorganisms. This project is performed together with collaborators and support from the Pacific Southwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases.