Multi-component solution phase reaction microarrays for proteomic applications
We have developed a novel microarray based proteomics platform technology that can rapidly assemble diverse multicomponent reactions without cross-contamination or the need for surface linkage. It can perform multiple on-chip enzymatic assays in the liquid phase, using biological fluids, at nanoliter volumes with minimal evaporation, minimal sample consumption and is highly reproducible. The proposed technology is compatible with standard microarraying and spot-analysis tools. Using glycerol-based buffers, we are able to microarray chemical compounds onto glass slides at 400 spots/cm 2 with each spot having a reaction volume of ∼1.6nL. Aerosol deposition allows metered delivery of biological samples, subsequent reagents and water into each reaction center in the entire microarray. The method can be utilized for both for homogenous fluorescence assays and label-free assays using MALDI-TOF Mass Spectrometry (MS). The technology platform is amenable to a variety of diverse proteomics applications such as profiling of protease-substrate interactions, complex protease mixtures such as blood, and high throughput screening reactions. ^ A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X = all natural amino acids except cysteine) to map protease-substrate interactions. Specificities of 19 serine proteases and 11 cysteine proteases were obtained from 129,960 separate microarray fluorogenic. ^ Using this library, we were also able to determine optimal substrates for various coagulation proteases, which overcame the cross-reactivity and non-specificity of currently available substrates. These optimal substrates were able to accurately determine the presence of different proteases from a complex mixture like plasma and can be potentially used for biomarker discovery, diagnosing various coagulopathies or functional phenotyping. ^ A potent cathepsin L inhibitor, MDL 28170, was identified from a 1000-compound inhibitor library using our method. Cathepsin L has been implicated in the mechanism of action for SARS-CoV membrane fusion and viral entry. Severe acute respiratory syndrome (SARS) is an acute respiratory illness caused by the SARS coronavirus (SARS-CoV). The compound MDL 28170 was able to potently and specifically inhibit pseudotype infection mediated by SARS-CoV and reduced live SARS-CoV replication. Additionally, a novel inhibitor of caspases 2, 4, and 6 was also identified from a 352-compound combinatorial library. ^
Engineering, Biomedical|Engineering, Chemical|Biophysics, General
"Multi-component solution phase reaction microarrays for proteomic applications"
(January 1, 2005).
Dissertations available from ProQuest.