COBRE: U KS: P2: STRUC CHAR GLYCOPROTEINS MASS SPEC; INFERTILITY, THYROID CANCE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。需要一种可靠的方法来表征糖蛋白，特别是当它们被用作药物时，以确保药物的安全和有效。然而，使用目前的技术，定性是耗时的，有时甚至是不可能的。这是因为目前的方案包括将碳水化合物从蛋白质中分离出来，并分别对这两个实体进行表征。因此，当存在多个糖基化位点时，这一过程导致不明确的结果，因为不清楚哪些碳水化合物来自哪个糖基化位点。这是研究糖蛋白激素hCG、hLH、hFSH和HTSH的关键障碍，所有这些激素都有多个糖基化位点。为了改进目前的表征方法，我们正在使用蛋白质水解液的组合来产生糖肽，然后用各种质谱学方法对糖肽进行分析。这些方法包括前体和产物离子扫描、MSN实验、金属衍生化策略，以及可能的在线分离/检测方法。此外，我们正在建立计算机算法来简化对光谱的解释。这项研究的预期结果将是一种有效的策略来表征存在多个糖基化位点的糖蛋白。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for 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.