Analysis of Folate Binding Protein N-linked Glycans by Mass Spectrometry
Department of Chemistry, University of Toledo , Toledo, Ohio 43606, United States. Journal of Proteome Research
(Impact Factor: 4.25).
12/2011; 11(3):1551-60. 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 (Hex(5-6)HexNAc(2)) as well as hybrid and complex N-linked glycans (NeuAc(0-1)Fuc(0-3)Hex(3-6)HexNAc(3-5)). The FBP from bovine milk contains putative structures corresponding to high-mannose (Hex(4-9)HexNAc(2)) as well as hybrid and complex N-linked glycans (Hex(3-6)HexNAc(3-6)), 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.
Available from: Christopher Taron
- "Prior reports have determined that several mammalian GPI-APs each possess experimentally verified combinations of N-and/or O-linked glycans    , however, the potential to which all GPI-APs may receive multiple types of glycosylation has not been systematically examined. Therefore, we computationally modeled and evaluated the human proteome for the presence of GPI-APs and their putative N-and O-linked glycan sites (Supporting Information Table 2). "
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ABSTRACT: GPI-anchored proteins (GPI-APs) are an important class of glycoproteins that are tethered to the surface of mammalian cells via the lipid glycosylphosphatidylinositol (GPI). GPI-APs have been implicated in many important cellular functions including cell adhesion, cell signaling, and immune regulation. Proteomic identification of mammalian GPI-APs en masse has been limited technically by poor sensitivity for these low abundance proteins and the use of methods that destroys cell integrity. Here we present methodology that permits identification of GPI-APs liberated directly from the surface of intact mammalian cells through exploitation of their appended glycans to enrich for these proteins ahead of LC-MS/MS analyses. We validate our approach in HeLa cells, identifying a greater number of GPI-APs from intact cells than has been previously identified from isolated HeLa membranes and a lipid raft preparation. We further apply our approach to define the cohort of endogenous GPI-APs that populate the distinct apical and basolateral membrane surfaces of polarized epithelial cell monolayers. Our approach provides a new method to achieve greater sensitivity in the identification of low abundance GPI-APs from the surface of live cells and the non-destructive nature of the method provides new opportunities for the temporal or spatial analysis of cellular GPI-AP expression and dynamics.This article is protected by copyright. All rights reserved
Proteomics 11/2014; 14(21-22). DOI:10.1002/pmic.201400148 · 3.81 Impact Factor
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ABSTRACT: This review is the seventh update of the original article published in 1999 on the application of MALDI mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2012. General aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, and fragmentation are covered in the first part of the review and applications to various structural types constitute the remainder. The main groups of compound are oligo- and poly-saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Much of this material is presented in tabular form. Also discussed are medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. © 2015 Wiley Periodicals, Inc. Mass Spec Rev.
© 2015 Wiley Periodicals, Inc.
Mass Spectrometry Reviews 03/2012; 31(2):183-311. DOI:10.1002/mas.20333 · 7.71 Impact Factor
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ABSTRACT: Most secreted and cell membrane proteins in mammals are glycosylated. Many of these glycoproteins are also prevalent in milk and play key roles in the biomodulatory properties of milk and ultimately in determining milk's nutritional quality. Although a significant amount of information exists on the types and roles of free oligosaccharides in milk, very little is known about the glycans associated with milk glycoproteins, in particular, the biological properties that are linked to their presence. The main glycoproteins found in bovine milk are lactoferrin, the immunoglobulins, glycomacropeptide, a glycopeptide derived from kappa-casein, and the glycoproteins of the milk fat globule membrane. Here we review the glycoproteins present in bovine milk, the information currently available on their glycosylation and the biological significance of their oligosaccharide chains.
Glycobiology 01/2014; 24(3). DOI:10.1093/glycob/cwt162 · 3.15 Impact Factor
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