Nitric oxide synthase products signal through interactions with heme centers in metalloproteins, such as guanylate cyclase and through covalent reactions with protein cysteine thiols. These nitrosylation reactions have broad relevance to eukaryotic cell biology in health and disease. However, nitrosylated proteins have often proven difficult to detect with the sensitivity and specificity needed for clinical and reseach samples. We are developing cavity ring-down spectroscopy to measure these protein modifications. We believe that this technology will dramatically improve both sensitivity and specificity it has the potential to will revolutionize the field. Here, we propose to design and fabricate an improved cavity ring-down instrument with a sample photolysis interface. This instrument will use advanced optical spectroscopy techniques to measure S- nitrosothiols in biological specimens. It will be used to studying a broad range of diseases and organ systems. It will be more powerful and more precise than previous technologies, measuring NO-modified proteins accurately in pM concentrations with minimal sample disruption. In the second Aim, it will also be applied to proteomic microarrays for rapid, high-throughput discovery.
S-nitrosylation chemistry has emerged as a major signaling pathway in biology. It is relevant to a broad range of cell signaling effects. Disorders of S-nitrosylation signaling have been identified in disease processes of virtually every organ system. However, detection of S-nitrosylation modifications in proteins has been fraught with difficulty. S-nitrosothiol bonds are artifactually made or lost during preparatory phases. Assays are commonly used near the limit of sensitivity. False positives and false negative signals are common. Metabolically labeled NO groups are challenging to follow with adequate sensitivity using mass spectrometry. We have been developing cavity ring-down spectroscopy as a novel method for detecting S-nitrosothiol bonds. It is at least 2 log orders more sensitive than currentl available techniques. In the current proposal, we will couple this technology with a photolysis assay platform and develop this system both for proteomic array analysis and for clinical sample assay. Samples can be isolated quickly with minimal preparatory steps and assayed sensitively. Importantly, 15NO-labeled modifications can be tracked for metabolic studies without the preparatory steps required for mass spectrometry. We believe this technology will revolutionize several emerging areas of biological research and medicine.