INTRACELLULAR MODIFICATIONS OF HUMAN APOLIPOPROTEIN E

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。人血浆载脂蛋白E（apoE）是负责清除脂蛋白残留，并已牵连到疾病，如家族性III型高脂蛋白血症和阿尔茨海默氏症？的疾病。已知其在合成后最初被唾液酸化，随后在血浆中被去唾液酸化。HepG 2细胞apoE的合成和细胞内修饰研究表明，修饰的apoE同工蛋白对应于具有2个、4个或6个唾液酸残基的唾液酸apoE形式。需要确认预期的O-糖基化位点，并通过质谱法表征修饰类型。从apoE的胰蛋白酶消化物中，可以通过LC-MS/MS在预期位置检测到一个二唾液酸化聚糖，然后也通过标准MALDI TOF MS发现。计划在凝集素的帮助下分离糖肽。振动冷却（VC）MALDI FT-MS可能有助于检测更高的唾液酸化，更不稳定的同系物。

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. Human plasma apolipoprotein E (apoE) is responsible for the clearance of lipoprotein remnants and has been implicated in diseases like familial type III hyperlipoproteinemia and Alzheimer?s disease. It is known to be initially sialylated after synthesis and later desialylated in the plasma. Studies of synthesis and intracellular modification of apoE by HepG2 cells have shown modified apoE isoproteins corresponding to the sialo apoE forms with two, four or six sialic acid residues. The expected O-glycosylation site needs to be confirmed and the kind of modification to be characterized by mass spectrometry. From a tryptic digest of apoE, one disialylated glycan could be detected at the expected position by LC-MS/MS, and was then also found by standard MALDI TOF MS. It is planned to isolate the glycopepitde(s) with the help of lectins. Vibrationally cooled (VC) MALDI FT-MS might help to detect higher sialylated, more instable homologues.