2-D Protein Biochips
“We have focused our 2-D protein fractionation platform on several different cancers using reverse-phase microarrays to look for new and more specific protein biomarkers using patient sera,” stated Timothy J. Barder, Ph.D., president of Eprogen.
Dr. Barder described Eprogen’s approach to biomarker discovery and validation as a two-stage process: first, applying the 2-D all-liquid phase fractionation strategy based on HPLC to solve the complex intact protein-expression analysis problem; and second, extending these fractionation strategies to produce 1-D and 2-D reverse-phase protein microarrays for probing with antibodies or autoantibodies.
To facilitate this separation technology, Dr. Barder’s team employs NPS® (nonporous silica) in an ultrafast, HPLC support for improved speed and resolution, tailored for proteins and mass spectrometry (MS). It provides a separation that is far superior to traditional methods such as 1-D gels, he reported.
Combining 2-D liquid-phase HPLC with microarray technologies opens up a range of possible strategies including screening multiple arrays produced from the same tissues with many different antibodies, or by analysis of autoantibody signatures or profiles (serological assay) for monitoring disease-progression analysis. This approach can also be applied to biomarker discovery and drug development studies, probing the 2-D arrays with well-documented patient serum samples.
Dr. Barder discussed the use of this approach in the investigation of prostate cancer biomarkers. David M. Lubman and his associates at the University of Michigan Medical School used a multidimensional liquid-phase protein fractionation of localized and metastatic prostate cancer tissue lysates to build protein microarrays, which were tested against serum from a cohort of 34 patients with either benign prostatic hyperplasia or clinically localized prostate cancer.
Spots on the microarrays that reacted positively were isolated and analyzed by MS. A number of proteins were detected that reacted against the autoantibodies from cancer patients’ serum, including 29 that were found to be prostate cancer specific; 12 of these were identified by single peptides. These may be proteins involved in pathway dysregulation that might otherwise be suppressed by the complexity of the cancer proteome. A larger melanoma study is currently under way using this approach to look for new serum markers for monitoring this cancer.
“The 2-D gel-drop microarrays can be highly automated, allowing maximum information from a given sample,” Dr. Barder concluded. “Many microarrays can be derived from a single fractionation and made available for testing with a variety of probes and detection methods.”
While protein microarrays are an engaging technology, they still present technical problems that may disarm the unwary. Their performance is dependent upon the antibodies used in the detection system, and conditions must be carefully optimized to ensure proper binding, and to eliminate nonspecific interactions. It should be cautioned that many antibodies don’t work well as capture reagents and bind poorly when reacted against cell extracts, even though they perform well under denaturing conditions and in Western blotting protocols.
There is a heightened awareness of these limitations within the biotech community, and the quality of data generated from protein microarrays is much more consistent and accurate than in the early days of this technology. These improvements should help to lower the high attrition rates of biomarker evaluation and allow the development of a new generation of biomarkers.