• Epitope mapping. Our Antigen-Specific Epitope-Targeting arrays will quickly and easily identify epitopes and enable rapid mapping antibody epitopes at the amino-acid level. Our approach will bypass the expensive cost of synthetic peptides, challenges of antigen purification, and the uncertainty of random peptide library screening. A nested series of deletion mutants, N-terminal, C-terminal, internal and tiling peptides can be generated rapidly and screened on ASET arrays at a low cost. Hundreds of arrays can be produced in one preparation and used for screening for many different antibodies to the target antigens. Our ASET arrays will allow epitope mapping in physiological buffers or under denaturing conditions.
• High-density arrays. Our high-density array development takes advantage of the mature semiconductor etching technology to produce silicon substrates with tens of thousands of nano-volume wells. Companion developments include a piezo-dispensing arrayer that allows the precise and accurate dispensation of genes into individual nano-wells, a filling device that allows the dispatch of nanoliter volumes of protein expression lysate into each well, and a sealing method that completely seals each micro-well to eliminate any well-to-well cross talk.

• High-sensitivity arrays. We have also been working with researchers at the company Meso Scale Discovery to reconfigure NAPPA to improve the sensitivity of signal detection. Meso Scale has created a new system that can detect signals using electrochemiluminescence, which means that the signal isn’t produced until stimulated by an electrochemical reaction. The benefit of this system is that it decreases background noise, and thus increases the signal to noise ratio, making it easier to detect binding of autoantibodies and differences between cases and control in NAPPA, for example. We are currently testing the NAPPA technology (i.e., spotting DNA and making protein in the Meso Scale wells) by spotting 4 spots of different DNA and detecting signals using electrochemiluminescence to see if the technologies are compatible. Future experiments will test if this system can be expanded for high throughput assays. If so, this system could significantly increase the sensitivity of our NAPPA assays to better identify biomarkers for disease and provide a potential tool for in the clinic assays.
• Surface Plasmon Resonance Imaging. Our lab has developed a high throughput approach that quantitatively characterizes more than 400 protein interactions simultaneously, collecting information on both interaction affinity and kinetics. In this platform, we have coupled our unique NAPPA protein microarray platform with SPR, enabling the assessment of affinities ranging from low micromolarity to high picomolarity. The SPRi method measures changes in refractive index very close to a sensor surface which are dependent on the dielectric properties of the metal, which in turn depend on what proteins are present on the surface. This method allows for real time and label-free detection of binding events. Coupled with NAPPA, the format has been used to characterize networks of interactions in a signaling pathway, including affinities and kinetics. This open format allows for testing the effects of post-translational modifications (PTMs) by testing interactions in their presence and absence.  It can also be used to explore the quantitative effects of numerous mutations and isoforms on the ability of a protein to interact with its targets.
• Protein interactions. We have developed a high-throughput unbiased protein-protein interaction screening platform on NAPPA microarrays displaying 10,000 human proteins and an independent validation platform on magnetic beads using fluorescein labeled detection reagents with high-affinity and high-specificity. With these tools, we are performing a series of proteomics studies to characterize bacterial pathogen/virus and host interactions and identify the novel host targets which may play important roles in cancers and infectious diseases.

Post-translational modifications. NAPPA can be coupled with a variety of biochemical modification systems to identify proteins that add post-translational modifications to other proteins or to identify protein targets. Furthermore, once developed, these systems provide an ideal method for screening drugs or other small molecule inhibitors. We have three main projects studying different post-translational modifications:

o   Phosphorylation and its applications in chronic myelogenous leukemia

o   AMPylation and its role in bacterial pathogenesis

o Citrullination and its role in the pathogenesis of rheumatoid arthritis

•  Sequence analysis – ACE. Designed to manage thousands of clones simultaneously, ACE uses an Oracle database to store information about clones at various completion stages, project processing parameters, and acceptance criteria. The software has been used successfully to evaluate over 250,000 clones at the LaBaer lab. Its use has allowed a dramatic reduction in the amount of time and labor required to evaluate clone sequences and has decreased the number of missed sequence discrepancies, which commonly occur during manual evaluation. In addition, ACE has helped to reduce the number of sequencing reads needed to achieve adequate coverage for making decisions on clones.
•  Sample tracking – FLEX. We have developed a laboratory information management system (LIMS) called FLEXGeneDB (FLEX) for tracking high throughput cloning processing, which is a major production pipeline in the lab. The FLEX system tracks every step in the production process and integrates with liquid handling robots to eliminate human errors. It maintains a detailed history for each plate and sample by capturing processing parameters, protocols and analytical results for the complete life cycle of the sample. FLEX is capable of managing clones produced outside our lab that require further processing such as transferring to different vectors or sequence validation. Visit the FLEX website.
• Advanced literature mining. High-throughput technologies, such as proteomic screening and DNA micro-arrays, produce vast amounts of data requiring comprehensive analytical methods to decipher the biologically relevant results. One approach would be to manually search the biomedical literature; however, this would be an arduous task. We developed two automated literature-mining tools, termed MedGene and BioGene, which can be used to comprehensively search the literature for associations between human genes and disease or biological themes, respectively.
• Next-generating sequencing. Our next generation sequencing service is now overseen by the ASU Genomics Facility.
• NAPPA tracking. The web-based NAPPA Tracking System manages key data and information throughout the NAPPA process including: sample management, slide production, experiment results, data analysis and visualization. The sample management module provides direct access to the DNASU Repository and FLEXgene database to access plasmids targeted for the NAPPA microarray platform. Processes to grow the clones on 96-well plates, store glycerol stocks on 96-well plates (for re-growth later), prepare and quantify DNA are tracked and detailed results recorded in the NAPPA relational database. The software is fully integrated with the DNA Factory to provide tracking of each step in DNA purification and quantification (see workflow of the DNA purification step below). The slide production module provides a visual tool to help researchers rapidly design and build high-density microarrays and generate the robot programs needed to produce these microarray slides in a high-throughput environment. The experiment results module helps scientists to design effective NAPPA experiments and to track high-throughput results. The final module provides powerful data analysis and visualization tools to enable researchers to effectively glean information from the vast data repository to help ensure high quality experiments and accelerate research.