STRUCTURAL ANALYSIS OF SULF2 PROCESSING OF EXTRACELLULAR HEPARAN SULFATE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 最近发现的Sulf 2酶用于修饰与细胞表面和细胞外基质蛋白聚糖相关的硫酸乙酰肝素（HS）。 这种重塑影响细胞对包括转化生长因子、血管内皮生长因子和成纤维细胞生长因子在内的生长因子家族的识别。 由于分析挑战，缺乏Sulf 2活性所需的HS结构域背景的详细结构信息。 这些信息对于理解Sulf 2酶作为组织位置和生长环境（发育和疾病）中时间变化的函数的功能作用至关重要。 Zaia等人开发了一种液相色谱-质谱（LC/MS）平台，用于分析硫酸肝素和其他糖胺聚糖类。 该平台包括亲水相互作用色谱和高分辨率电喷雾质谱。 四链体聚糖稳定同位素标签用于促进HS寡糖的定量。 该平台是评估Sulf 2酶活性的理想平台。 将检测Sulf 2活性与来自MSR可用的几种器官的HS。 HS样品将用Sulf 2消化。 然后使用肝素裂解酶对样品进行部分解聚，以产生从二聚体到十聚体的寡糖。 将使用四链体聚糖标签对寡糖进行衍生化，以便于定量。 将使用LC-LTQ-Orbitrap MS系统通过高精度质量测量测定寡糖组成。 将使用串联质谱法分析观察到的丰度发生变化的寡核苷酸。 产物离子模式将用于确定HS寡糖结构。 预计准确的质量测量将确定寡糖来源的HS结构域（N-硫酸化、N-乙酰化或杂合）。 丰度的变化将决定Sulf 2作为哺乳动物器官功能的总体结构域偏好。 串联MS将提供有关Sulf 2作用位点的其他结构细节。

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. The recently discovered Sulf2 enzyme acts to modify heparan sulfate (HS) associated with cell surface and extracellular matrix proteoglycans. This remodeling affects cellular recognition of growth factor families including transforming growth factors, vascular endothelial growth factors, and fibroblast growth factors. Detailed structural information on the HS domain context required for Sulf2 activity is lacking due to analytical challenges. Such information is essential to understanding the functional roles of Sulf2 enzymes as a function of tissue location and temporal changes in the growth environment (development and disease). Zaia et al have developed a liquid chromatography-mass spectrometric (LC/MS) platform for analysis of heparin sulfates and other glycosaminoglycan classes. This platform includes hydrophilic interaction chromatography and high resolution electrospray mass spectrometry. A tetraplex glycan stable isotope tag is used to facilitate quantification of HS oligosaccharides. This platform is ideal for assessing the activity of Sulf2 enzymes. Sulf2 activity will be tested versus HS derived from several organs available at the MSR. HS samples will be digested with Sulf2. Samples will then be partially depolymerized using heparin lyases so as to produce oligosaccharides ranging from dimers to decamers. The oligosaccharides will be derivatized using tetraplex glycan tags to facilitate quantification. The oligosaccharide compositions will be determined by high accuracy mass measurement using an LC-LTQ-Orbitrap MS system. Oligosaccharides observed to undergo changes in abundance will be analyzed using tandem mass spectrometry. The product ion pattern will be used to determine HS oligosaccharide structures. It is expected that accurate mass measurement will define the HS domain (N-sulfated, N-acetylated, or hybrid) from which the oligosaccharide derives. Changes in abundance will determine the overall domain preference of Sulf2 as a function of mammalian organ. Tandem MS will provide additional structural detail regarding the sites acted upon by Sulf2.