The N-acetylglucosamine (O-GlcNAc) modification is a reversible attachment of O-GlcNAc onto serine or threonine residues of intracellular proteins. This modification mediates cellular activities by regulating protein trafficking, conformational change, and by antagonizing phosphorylation. Many human pathologies exhibit aberrant O-GlcNAcylation of specific proteins. Current strategies for detecting O-GlcNAc, however, are insufficient. Immunoblotting and mass spectrometry are the state-of-the-art methods to study protein O-GlcNAcylation. To detect the modification of a specific protein, Western blots are typically paired with a prior precipitation step with either target specific antibodies or anti-O-GlcNAc affinity reagents. This enrichment process can be labor intensive and lead to major sample loss due to the relatively low affinity of lectins and anti-O-GlcNAc antibodies. Mass spectrometry is the most widely used tool for profiling protein-specific O-GlcNAc modification on a global level. The generation of high-quality glycoproteomics data, however, requires large quantities of sample followed by rigorous and labor intensive enrichment to ensure adequate representation of low-abundance glycoproteins. Also, the instrumentation to perform these analyses is possessed by only a few labs due to it being very expensive and requiring a high level of specialized expertise to operate. We herein propose to develop a low-tech, broadly accessible method for analyzing the O-GlcNAcylation state of specific proteins. Changes in O-GlcNAcylation are mechanistically implicated in many human diseases and thus represent a fertile ground for scientific investigation. The broad goal of this proposal is to develop and disseminate an accessible PCR-based glycoproteomics platform for monitoring changes in O-GlcNAcylation. We describe the development of a technique that will allow the detection of protein-specific glycosylation directly from lysate using chemical probes of O-GlcNAc in tandem with multiplexed proximity ligation assays and fluorescence-based quantitative PCR.
Certain proteins called transcription factors are responsible for turning genes 'on' and 'off', and these proteins need to be activated and also directed to the target gene to perform their duty. It is suspected that a change in the protein's state, termed its O-GlcNAcylation state, may govern the activities of these transcription factors, but the O-GlcNAcylation state of a protein currently can only be analyzed by very long procedures, and by very few scientists who have highly specialized and expensive equipment. We propose herein to develop a low- tech methodology which will enable the broader scientific community to easily analyze O-GlcNAcylation state; this will broadly impact scientific and biomedical endeavors wherein gene turn-on is of interest - from stem cell therapies to the transition of cancer cells from benign to malignant.
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