Due to the complexity of the human proteome, large dynamic range in protein concentrations, and changes of protein abundance due to minor physiological stimuli, processing of samples for proteomic analysis is a challenging task.
“Over a period of 10 years, we have systematically developed methods for protein extraction from tissues that provide maximum protein solubilization and preserve protein native conformation,” says Joerg Hoheisel, Ph.D., head of functional genome analysis, German Cancer Research Center.
“We found that depletion of high-abundance proteins and other types of fractionation led to co-depletion of minor proteins and, therefore, introduced a strong bias in protein representation,” explains Dr. Hoheisel, one of the key presenters at the U.S. HUPO Conference on the Future of Proteomics in March in San Francisco.
“Our goal was to create a set of reproducible conditions for protein analysis by antibody arrays using unfractionated body liquids and tissue samples. An entirely new protein purification and hybridization strategy had to be worked out for the antibody arrays.”
Once the appropriate conditions were optimized, Dr. Hoheisel’s group was able to establish distinct pancreatic cancer signatures by analyzing nearly 900 samples. Another signature forecasts the recurrence of bladder cancer.
Similar to cDNA microarrays, antibody arrays are analyzed by two-color fluorescent assay. Moreover, in certain conditions only one dye molecule binds one protein molecule. The team adapted already existing single-molecule detection techniques to visualize individual affinity capture events.
Selective illumination excites fluorophores only in a small region of the specimen immediately adjacent to the glass-water interface. Such a readout allows for easier discrimination between fluorescence arising from captured molecules and background caused by light scattering at the surface.
Using this approach, as few as 600 molecules per spot could be detected and counted. The team applied this method to develop a proof-of-concept diagnosis of tuberculosis based on capture and detection of lipoarabinomannan, a polysaccharide marker of tuberculosis. Current tuberculosis diagnosis is not sensitive enough to detect the infection at early stages when the treatment may be the most effective.
With the rapid re-emergence of tuberculosis worldwide, a reliable and sensitive diagnosis is of critical importance for disease control. Antibody capture combined with fluorescence detection seems to be well positioned to address this growing need.