Microarrays have advanced significantly over the years, which is a good thing as genomics would not exist if not for that progress. Protein microarray development, however, has not seen as much success as DNA microarray development, which has been somewhat worrisome for proteomic scientists—and life scientists, in general. In fact, it is likely that the genomics revolution may come to grinding halt if protein microarray development does not catch up soon.
If the focus on protein microarrays at many of this year’s scientific conferences is any indication, academic and industry researchers are aware of the dire need for progress in the field and are endeavoring to deliver the cutting-edge methodologies and tools that will help to avert a crisis.
Yashu Liu, Ph.D., a postdoctoral fellow at the University of Michigan (UM), spoke at “PittCon” about development of a protein microarray to perform biomarker screening in serum from hepatocellular carcinoma (HCC) patients. From this screen, UM researchers identified five biomarker candidates (seroplasmin, Histidine-rich glycoprotein, Complement C3, CD14, and hepatocyte growth factor) that were initially discovered using mass spectrometry and then validated using the protein array.
To create the array, Dr. Liu raised monoclonal antibodies against known serum proteins and bound them on a nitrocellulose-coated glass slide. The screen was then performed by incubating the array with HCC patient serum to screen for potential HCC biomarkers (antigens).
Biotinylated lectins were then used to detect glycan structures on the antigens in the serum, followed by incubation with a fluorescent dye to detect the biotin. “We developed this method so that people could use it with any kind of cancer. If you think there will be a glycan structure change in cancer antigens, then you can apply this process to another type of cancer or another disease,” said Dr. Liu. “Alphafeto protein is not good enough for early detection of HCC so the proteins we found could potentially aid in the detection and diagnosis of this cancer.”
Matthew Linman, a graduate student in Quan Cheng, Ph.D.’s research group at the University of California, Riverside, reviewed currently unpublished data on the initial development of a lipid microarray designed to immobilize transmembrane proteins at “Pittcon”. SPR was used as a functional assay for proteins immobilized on the array. The array, which is based on a calcinated chip, consists of a thin layer of glass atop a layer of gold.
There are several reasons for this design. One reason is that, in order for SPR to work, a gold layer is required. Lipid vesicles, however, do not fuse directly to gold but will fuse to glass. By combining both elements in one array, the group was able to achieve lipid fusion and successful in situ protein function results via SPR.
“We are currently designing the surface, which is called a biological interface, to mobilize epidermal growth factor receptor (EGFR),” said Linman. “The idea behind using this array is to create a surface to test out small molecule inhibitors of EGFR to give patients with head and neck cancer more options than just monoclonal antibody-based therapies such as Erbitux.”
For the last five years, Peter Nilsson, Ph.D., associate professor at KTH Royal Institute of Technology, has been developing various types of protein arrays. Dr. Nilsson’s lab has produced two kinds of protein arrays—antigen arrays for antibody validation, as well as suspension arrays, which are color-coded beads with immobilized antibodies, to perform plasma profiling.
“Our goal is to take all of the antibodies that come out of the Human Protein Atlas project and profile them on serum or plasma cohorts,” said Dr. Nilsson. “We increased the number of antibodies and samples that can be used for simultaneous profiling by direct-labeling assay based on a biotin-streptavidin interaction.”
The main purpose of this array is to discover new biomarkers by comparing plasma and serum samples from various disease cohorts. Dr. Nilsson will present a massive number of data points generated with antibodies profiled on the samples at Select Biosciences’ “Advances in Microarray Technology” to be held in Dublin next month. His presentation will show the “possibility of using a large number of antigens and antibodies to identify biomarkers of disease in a discovery context.”