基质辅助激光解吸电离飞行时间质谱技术研究人血清转铁蛋白稳定性及裂解产物

制备质谱纯人血清转铁蛋白(HTF),供分子结构分析。选用SDS-PAGE、胶外酶解、基质辅助激光解吸/电离质谱技术(MALDI-TOF)、数据库检索和比对技术鉴定铁饱和HTF、双铁HTF(HTF-2Fe3 )、单铁HTF(HTF-Fe3 )和脱铁HTF(apoHTF)的稳定性和裂解产物。以乙腈溶液作为洗脱相,发现铁饱和HTF在RP-HPLC分离纯化过程中产生裂解现象。铁饱和HTF和HTF-2Fe3 经乙腈处理后均能产生不同分子量的短肽裂解产物,指出HTF结构稳定性与络合铁离子数量有关。铁组分改善了HTF分子结构的稳定性。采用比对法,研究在乙腈作用下HTF裂解成为各种各样短肽的规律,初步阐明其裂解机理。在乙腈作用下,HTF可能通过蛋白质去折叠途径,形成不同多聚态HTF或多肽裂解产物。推测目前用于临床诊断先天性糖基化紊乱(CDG)和慢性酒精滥用(CAA)疾病低准确率的起因可能是受HTF裂解产物或多聚体的干扰。

Stability and Splitting Produces Revealed by Matrix-Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry in Human Serum Transferrin

Human serum transferrin (HTF) with purity of mass spectrometry were prepared for the analysis of molecular structure. Here, several analytical techniques, such as SDS-PAGE, enzymolysis out-gel, MALDI-TOF mass spectrometry, database search, and comparison approach were employed to identify the stability and splitting produces among iron-saturated HTF, HTF-2Fe3 , HTF-Fe3 and apoHTF. Using acetonitrile as a component of eluant for RP-HPLC separation, it was found that the eluant enabled to decompose HTF into peptides, which indicated that the structural stability of HTF was tightly relative to the iron numbers binding to the protein, and the iron played an important role in improving the stability. The regulation and pathway of forming various peptides from the HTF treated with acetonitrile directly, were studied for revealing the splitting mechanism. The acetonitrile resulted in the unfolding of HTF for forming various different polymers splitting products of the protein. It could be presumed that the reason for the low reliability of diagnosis both congenital disorder of glycosylation and chronic alcohol abuse in clinic is the interferential factors of aplittying products in HTF.