Proteomics is a powerful approach for discovery of molecular targets for disease therapeutics and novel biomarkers for disease diagnosis. Mass spectrometry (MS) and protein array are key technologies for proteomics because they provide exquisite sensitivity and high throughput for protein/peptide measurement. Our primary interests in the proteomics field lie in the development and application of novel proteomics technologies to studying human diseases including oral diseases.
(1) Oral cancer proteomics
SCC of the oral cavity and oropharynx is the sixth most common human cancer. In spite of advances in surgery, radiation, and chemotherapy, the overall 5-year survival rates for oral cancer have remained very low for the past decades and has remain among the worst of all cancer death rates. The high morbidity rate of oral cancer can be attributed to factors including non-responsiveness to chemotherapy and radiation therapy, late presentation of the lesions and the lack of biological markers for the early detection of these lesions. It is hoped that a better understanding of the molecular determinants of oral carcinogenesis will improve diagnosis, treatment, and monitoring of the disease.
Quantitative proteomics using MS with stable isotope labeling is an emerging high-throughput technology for relative measurement of protein levels, in a global fashion. Currently we are combining laser capture microdissection (LCM) and quantitative proteomics to conduct quantitative analysis of oral cancer tissue. Protein samples form LCM-dissected cancer and control tissue cells are labeled with iTRAQ reagents and then analyzed by LC-MS/MS. A number of up- and down-regulated proteins have been identified, including tumor- or cell cycle-associated ones. These proteins hold the potential to be diagnostic biomarkers and/or therapeutic targets of this specific cancer.
(2) Human salivary proteome project (HSPP)
Human saliva is a mixture of gingival crevicular fluid and the secretion of parotid, submandibular, sublingual and minor salivary glands. Human saliva contains arrays of proteins that have distinct biological functions. For instance, salivary mucins form coating on oral tissues and protect oral mucosa. Statherin and proline rich proteins prevent precipitation or crystallization of calcium phosphate salts. Amylase and lingual lipase maintain the digestive function of saliva. Lysozyme, lactoferrin, peroxidase and immunoglobulins constitute the oral defense system to protect the oral cavity by modulating microbial colonization and metabolism. Due to their critical roles in maintaining the health of oral cavity, it is very important to understand the protein composition of saliva as well as the biological functions of those proteins.
The HSPP is a multi-institutional multidisciplinary research consortium aiming to generate a complete catalogue of all salivary secretory proteins using state of the art, sensitive and high-throughput proteomic technologies. We are currently utilizing both bottom-up and top-down proteomics approaches to identify proteins in human saliva, including their stratification according to their parotid, submandibular and sublingual origins. Using both 2-D gel electrophoresis/MS and “shotgun” proteomics, we have identified around 600 proteins, which represents the most comprehensive catalogue of whole saliva proteins up to date. By performing proteomics and anatomy studies, we have also discovered that submandibular and sublingual secretions may be segregatable in certain human population. For further details about HSPP, please refer to <http://www.hspp.ucla.edu/>.
(3) Discovery of saliva protein biomarkers for human diseases
Human saliva is attractive for disease diagnostics because its collection is less invasive than that of blood for serum/plasma analyses and because blood concentrations of many components are indeed reflected in saliva. This bio-fluid has been proven to be very valuable for diagnosis of human diseases such as HIV, periodontal diseases and hepatitis. We have demonstrated an integrated proteomic approach to discover saliva peptide/protein biomarkers for human oral cancer. By MALDI-MS profiling of saliva samples and subsequent statistical analysis, a panel of saliva peptides/proteins have been revealed at significantly differential levels between cancer/control groups. We have also established a straightforward strategy by using LC fractionation, MALDI-MS, LC-MS/MS and nanospray MS/MS to identify candidate biomarkers. The discovery may lead to future application of using saliva and saliva peptides/proteins for oral cancer diagnosis. Currently we are also utilizing MALDI-MS and 2-D gel electrophoresis for profiling of saliva samples from Sjogren Syndrome patients. Saliva peptide/protein biomarkers may be discovered for diagnosis of this autoimmune disease.
(4) Lectin protein array
Aberrant glycosylation of proteins occurs in all types of human cancers and many glycosyl epitopes constitute tumor-associated antigens. We are currently developing lectin array technology for high-throughput mass spectrometric analysis of glycoproteins associated with human cancers. Specifically, we array a panel of lectins on chemically modified surface to capture glycoproteins in serum/tissue samples and subsequently mass spectrometry techniques are used to detect and identify those glycoproteins. The developed lectin array technology will have great potential in harnessing the glycoprotein alterations during the process of carcinogenesis and discovering glycoprotein signatures for cancer detection.
UO1- DE016275-01 (Wong)
Human Salivary Proteome
UCLA New Faculty Seed Grant (Hu)
UCLA School of Dentistry
Quantitative saliva proteome analysis: Discovery of saliva protein markers for oral cancer
1. Hu, S, Xie, Y, Ramachandran, P, Loo, R R, Li, Y, Loo, J A, and Wong, D T. Large-scale identification of human oral fluid proteins by liquid chromatography-mass spectrometry. Proteomics, 2005, 5(6):1714-28.
2. Hu, S, Denny, P, Xie, Y, Loo, J A, Wolvinsky, L E, Li, Y, McBride, J, Ogorzalek, Loo, R R, Navazesh, M, and Wong, D T. Differentially expressed protein markers in human submandibular and sublingual secretions. Int J Oncol, 2004, 25: 1423-1430.