Analysis of Folate Binding Protein and Associated N-Glycans by Mass Spectrometry and Light Microscopy

by Nidhi Jaiswal

Institution: University of Toledo
Department: Chemistry
Degree: MS
Year: 2011
Keywords: Analytical Chemistry; Chemistry; Folate Binding Protein; Mass Spectroscopy; N-Glycans; Light Microscopy
Record ID: 1929379
Full text PDF: http://rave.ohiolink.edu/etdc/view?acc_num=toledo1302210585


Folate binding protein (FBP), also known as folate receptor (FR), is a glycoprotein which binds vitamin folic acid and its analogues. The FBP contains multiple N-glycosylation sites and is overexpressed in human cancers including ovarian, lung, kidney, and breast cancer. However, the structure and the composition of N-glycans bound to the FBP are still unknown. We performed structural characterization of FBP N-linked glycans originating from bovine and human milk. The N-linked glycans were enzymatically released from FBP, purified, and permethylated. The glycans were analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their further structural characterization. In addition, deglycosylated FBP was purified by solid phase extraction and analyzed by MALDI-MS. It was found that FBP from human milk contains putative structures that have composition consistent with high-mannose (Hex5-6HexNAc2) as well as hybrid and complex N-linked glycans (NeuAc0-1Fuc0-3Hex3-6HexNAc3-5). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex4-9HexNAc2) as well as hybrid and complex N-linked glycans (Hex3-6HexNAc3-7), but these glycans mostly do not contain fucose and sialic acid. To image the FBP in live and fixed cells, KB cells were incubated with folic acid conjugated with fluorescein isothiocyanate (FITC) and FITC alone. Cells labeled with conjugated folic acid showed higher fluorescence intensity than cells labeled with FITC. In addition, KB cells were cultured in order to isolate FBP from cell lysate and cell culture media using an epoxy-activated folate-sepharose column. In summary, glycomic characterization of FBP provided a valuable insight into the structure of this cancer-relevant glycoprotein, and may be beneficial for a glycomic analysis of FBP originating from diseased cells and tissues. Imaging of FBP in cells can help to locate folate-based anti-cancer drugs and to follow their effect on cancer cells.