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With Aid of Mass Specs, HSPP Hopes to Move Saliva into Clinical Testing Arena

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Despite its accessibility, saliva has traditionally played a secondary role to blood and urine in the clinical testing arena. But scientists involved in the half-year-old Human Salivary Proteome Project say that is about to change, thanks to new technology that allows for greater protein detection sensitivity, and new support and funding from the National Institutes of Health.

According to David Wong, director of the University of California Los Angeles’ division of the HSPP, over the past two years the National Institute of Dental and Craniofacial Research has invested $57 million for salivary research that involves proteomics.

The grant money is helping to build nanotechnology-based biosensors that can eventually be used for saliva tests, and it is helping to uncover the salivary proteome, which will be used as a basis for diagnostic tests.

“The reason the NIH is investing all these resources into saliva is they want to see it moving into the real world,” said Wong, who is also the associate dean of research at UCLA’s School of Dentistry. “They want to truly investigate the diagnostic potential of saliva, and to create a national initiative to catalyze this concept into reality.”

The NIH believes proteomics, especially mass spectrometry, can play a role in this endeavor, Wong added. The agency issued several RFA’s for proteomic salivary research, which led to the founding of the HSPP last September.

Three research groups were awarded about $1 million per year for four years to investigate the salivary proteome. The groups are at UCLA and the University of Southern California, the University of California San Francisco, and the Scripps Institute.

The goal of the HSPP is to collect and distribute saliva from five ethnic groups, and to use both bottom-up and top-down proteomic approaches to decipher the complete salivary proteome and complexes associated with the proteome. Biocomputational and bioinformatics approaches will be used to catalogue and annotate the salivary proteome, and to create an HSPP database called the Salivary Proteome Knowledge Base.

To date, the HSPP has catalogued about 330 proteins in the SPKB. Members of the initiative are investigating whether mass-spec-based saliva testing can help diagnose oral cancer, early onset diabetes, Alzheimer’s disease, ovarian cancer, and pancreatic cancer.

Salivary diagnostics have been around for a long time, Wong noted, but saliva-based tests have not been popular in part because older technologies, such as ELISA tests, have not been sensitive enough to detect the relatively low amounts of potential diagnostic analytes in the fluid.

“Saliva just hasn’t been the darling of the medical profession,” said Paul Denny, a professor of diagnostic sciences at the University of Southern California School of Dentistry who is heading up the functional proteomics arm of the HSPP. “If you’ve ever been to China, spit there has a negative connotation. Some people actually believe that it harbors bad toxins, so the more you spit, the more you get rid of the toxins.”

But with the advent of sensitive mass spectrometers that can detect very low concentrations of proteins, saliva makes sense in the clinical world as a fluid that can be used in non-invasive “home tests.”

“Instead of doing a battery of tests where your blood chemistry falls between some normal range, saliva could be a totally non-invasive substitute,” Wong said. “Oral fluid could be a wellness fluid — an indicator of whether or not someone is in good health.”

For the purposes of this research, saliva is collected from each of the three major salivary glands: the parotid gland, the submandibular gland, and the sublingual gland. The saliva from each gland is kept separate in order to make it easier to analyze the protein composition from each gland, said Lawrence Wolinsky, the director of clinical research at the UCLA School of Dentistry who is heading up the clinical core group of the HSPP.

“Saliva is very heterogeneous. There are a lot of things in there — bacterial products, food products, cells decimated off mucosal tissues, substituents from the oral pharynx region,” he said. “From a logical standpoint, if you want to know the protein composition from the major salivary glands, the most logical way to go is to eliminate all of those other factors by collecting directly from each gland,” Wolinsky explained.

Wolinsky noted, however, that while saliva is being collected directly from ducts for the HSPP, in the future, he envisions whole saliva being used for diagnostic tests.

“We’re looking at something like the AIDS test where you spit in a cup and you have a dipstick,” he said.

In terms of proteomic analysis, saliva, like serum, is challenging because there is a large range in concentrations of protein in the fluid, said Julian Whitelegge, an associate professor of psychiatry at UCLA who is directing the top-down proteomics group for the HSPP. Whitelegge is dealing with the complexity of the salivary proteome by doing two-dimensional high-performance liquid chromatography on samples before submitting them into a tandem mass spectrometer in his lab.

Whitelegge’s specific project is to separate out proteins that are deliberately secreted into saliva from proteins that are accidentally sloughed off, or spilled out of ruptured cells into the fluid.

“We’ve been funded [by the NIH] to define the secreted proteome,” Whitelegge said. “The onus is on us to find out what is deliberately secreted, and what is sloughed off as background.”

Other HSPP groups are actively looking for disease biomarkers within the salivary proteome. Wong’s group published a paper in Clinical Cancer last December on an RNA biomarker that can 90 percent of the time correctly distinguish patients that have oral cancer from normal subjects.

“It was very serendipitous,” said Wong. “We were studying the salivary proteome when we came upon this RNA biomarker. Our clinical data tells us that it is unusually sensitive and specific.”

Wong said that his research group will continue to study the salivary proteome, as well as the salivary transcriptome. Aside from oral cancer, the group is also working on finding biomarkers for early onset diabetes, Alzheimer’s disease, ovarian cancer and pancreatic cancer.

Denny said that the range of diseases for which biomarkers can be found in saliva is as broad as the range of serum biomarkers. “Pretty much anything that’s in serum is in saliva,” he said. “In some cases it may be present in about the same amount, in other cases it may be a lot less. In early days, being present at a lot less amount was a problem, but nowadays, sensitivity is no longer an issue.”

More information about the UCLA/USC HSPP group can be found at http://www.hspp.ucla.edu. The UCSF and Scripps Institute HSPP groups do not yet have websites.

— TSL

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