This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.A reliable method to characterize glycoproteins is needed, particularly when they are used as pharmaceuticals, to assure that drugs are safe and effective. Yet characterization is time-consuming, and sometimes impossible, using the current technologies. This is because current protocols involve cleaving the carbohydrate from the protein and characterizing the two entities separately. Thus, when multiple glycosylation sites are present, this process leads to ambiguous results, as it is unclear which carbohydrates came from which glycosylation site. This is a critical obstacle in studying the glycoprotein hormones hCG, hLH, hFSH, and hTSH, all of which have multiple glycosylation sites. To improve upon current characterization methods, we are using a combination of proteolytic digestions to produce glycopeptides, followed by analysis of the glycopeptides by various mass spectrometric methods. The methods include precursor and product ion scanning, MSn experiments, metal-derivatization strategies, and possibly, on-line separation/detection methods. In addition, we are building computer algorithms to simplify the interpretation of the spectra. The expected outcome of the research will be an effective strategy to characterize glycoproteins with multiple glycosylation sites present.
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