Analysis of Folate Binding Protein N-linked Glycans by Mass Spectrometry

• Published in: Journal of proteome research 2012-03, Vol.11 (3), p.1551-1560
• Main Authors: Jaiswal, Nidhi, Saraswat, Suraj, Ratnam, Manohar, Isailovic, Dragan
• Format: Article
• Language: English
• Subjects: Animals; Carbohydrate Sequence; Cattle; Congenital Disorders of Glycosylation; Folate Receptors, GPI-Anchored - chemistry; Humans; Milk - chemistry; Milk Proteins - chemistry; Milk, Human - chemistry; Molecular Sequence Data; Molecular Weight; Polysaccharides - chemistry; Polysaccharides - isolation & purification; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr2006044

The folate binding protein (FBP), also known as the folate receptor (FR), is a glycoprotein which binds the vitamin folic acid and its analogues. FBP contains multiple N-glycosilation sites, is selectively expressed in tissues and body fluids, and mediates targeted therapies in cancer and inflammatory diseases. Much remains to be understood about the structure, composition, and the tissue specificities of N-glycans bound to FBP. Here, we performed structural characterization of N-linked glycans originating from bovine and human milk FBPs. The N-linked glycans were enzymatically released from FBPs, purified, and permethylated. Native and permethylated glycans were further analyzed by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry (MS), while tandem MS (MS/MS) was used for their structural characterization. The assignment of putative glycan structures from MS and MS/MS data was achieved using Functional Glycomics glycan database and SimGlycan software, respectively. 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 (Hex3–6HexNAc3–6), but these glycans mostly do not contain fucose and sialic acid. Glycomic characterization of FBP provides valuable insight into the structure of this pharmacologically important glycoprotein and may have utility in tissue-selective drug targeting and as a biomarker.