Someday, an instrument found in a medicine cabinet might be able to detect disease by scanning for biomarkers in saliva. But first, Joseph Loo, who is part of the University of California, Los Angeles, arm of the salivary proteome project, and his colleagues need to put the finishing touches on their paper characterizing protein components found in healthy people’s saliva.
“The NIH, when they constructed this RFA, they did it in a logical or a step-wise fashion,” says Loo, a biochemistry professor. “They rationalized that one needed to have a catalogue of all the proteins from a healthy individual first before one sort of dives into looking for protein markers for disease.” The team has identified between 1,200 and 1,500 unique salivary proteins so far.
To create that catalogue, the researchers collected saliva samples — a process that involved more than spitting into a cup. Once a month, Loo’s dental school collaborators collected whole saliva and samples directly from the different salivary glands of 10 healthy people.
Samples in hand, Loo and his lab threw nearly every proteomics tool there is at the saliva. At first, they used one- and two-dimensional gel electrophoresis to visualize the bands or spots and then punched them out to identify the proteins by mass spectrometry. Eventually, they explored other ways to fractionate the complex samples, including HPLC, two-dimensional chromatography, and iso-electric focusing. “Really, we’ve sort of employed the entire kitchen sink of everything to fractionate proteins,” Loo says.
In addition to identifying and characterizing the proteins, the Loo lab set up the Salivary Proteome Knowledge Base, a database containing information about the proteins and peptides found by the labs involved in the project. Also, the team created a wiki in part to receive feedback about the catalogued proteins from outside researchers. “People have told us, ‘Oh, I didn’t know this protein was in saliva’ or ‘I knew that protein was in saliva for 50 years, but you missed the phosphorylation site,’” Loo says.
With their techniques refined, the crew is starting to look into disease biomarkers. They are particularly interested in cancer markers and are working on collaborating to research oral cancer, as well as lung or breast cancer.
After the markers are sorted out, the spit-as-diagnostic tool might become a reality. The NIH funded a sister project to engineer a tool to detect the markers unearthed by the proteomics teams. “Once people know what markers to find for a specific disease, then you’d better have a device that could be used to measure for these markers,” Loo says. Many of these forward-looking devices are nearly ready. “They’re just looking for input now,” he says. And he’s almost done with the first step to give them the data they need.