High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid

Anumula, Kalyan R.; Dhume, Shirish T.

doi:10.1093/glycob/8.7.685

Cited by 261 publications

(231 citation statements)

References 21 publications

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“…Another strategy of carbohydrate analysis was performed by deglycosylation using N-glycanase digestion or hydrazinolysis. The sugar components were derivatized with 2-aminobenzamide and sodium borohydride (12). The derivatives were purified by reversed phase HPLC for analysis by mass spectrometry.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Alzheimer's β-Secretase Protein BACE

Haniu

Denis

Young

et al. 2000

Journal of Biological Chemistry

210

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The cerebral deposition of amyloid ␤-peptide is an early and critical feature of Alzheimer's disease. Amyloid ␤-peptide is released from the amyloid precursor protein by the sequential action of two proteases, ␤-secretase and ␥-secretase, and these proteases are prime targets for therapeutic intervention. We have recently cloned a novel aspartic protease, BACE, with all the known properties of ␤-secretase. Here we demonstrate that BACE is an N-glycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We have used a se- , and Cys 330 -Cys 380 ). Despite the conservation of the active site residues and the 30 -37% amino acid homology with known aspartic proteases, the disulfide motif is fundamentally different from that of other aspartic proteases. This difference may affect the substrate specificity of the enzyme. Taken together, both the presence of a transmembrane domain and the unusual disulfide bond structure lead us to conclude that BACE is an atypical pepsin family member.The hallmarks of Alzheimer's disease (AD) 1 pathology are brain plaques and vascular deposits (1) consisting of the 4-kDa amyloid ␤-peptide (A␤) (2). Overproduction of the 42-amino acid form of A␤, A␤42, has been suggested to be the cause of all known cases of familial early onset AD (3), and it is assumed that A␤42 deposition plays an early and critical role in sporadic AD as well. Therefore, A␤ metabolism has attracted considerable interest. In 1987 it was shown (4) that formation of A␤ requires proteolytic cleavage of a large type I transmembrane protein, the ␤-amyloid precursor protein (APP), which is constitutively expressed in most cell types. Over the next decade the proteolytic processing of APP has been studied in great detail in a variety of systems by many groups. Taken together, these studies have shown that A␤ is generated at a low rate by most cells analyzed and that two different proteolytic activities are required for A␤ generation. First, ␤-secretase cleaves APP to generate the N terminus of A␤, and second, ␥-secretase cleaves the C terminus, leading to the release of A␤ (for review see Ref. 5). Studies with intact cells expressing APP and the endogenous secretases have led to conclusions about the properties of the ␤-and ␥-secretases, e.g. their tissue distribution, subcellular localization, substrate requirements (see e.g. Ref. 6) etc., but until recently the identity of both ␤-and ␥-secretase was unknown. This changed when we very recently identified the novel transmembrane aspartic protease BACE as the major ␤-secretase (7). Three subsequently published independent studies (8 -10) have confirmed this conclusion. Here we characterize the BACE protein. We show that BACE is an Nglycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We determine the processing and N-glycosylation sites and the disulfide bonds. Our results demonstrate that BACE is an unusual member of the pepsin family. EXPERIMENTAL PROCEDURESMat...

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Section: Methodsmentioning

confidence: 99%

Characterization of Alzheimer's β-Secretase Protein BACE

Haniu

Denis

Young

et al. 2000

Journal of Biological Chemistry

210

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“…63 Polyamide-based normal-phase matrices have been introduced for the isolation of small amounts of labeled oligosaccharides. 64 The polyamide formats for this purpose include mini sample preparation columns and filters (e.g., Discovery DPA-6S ® , 65 Speed Amide-2/Polyamide 6S ® , and nylon filter 64 ). Other hydrophilic phases, such as glass, cellulose, and CN-silica, are also used for removing excess reagents.…”

Section: Specific Entrapment Of Glycansmentioning

confidence: 99%

“…Isomeric oligosaccharides can be separated using shallow gradients. 64,[109][110][111][112][113] High resolution of oligosaccharides labeled with 2-AA 64 and 2-AP 114 has been obtained using Asahipak NH2P-50 columns. In this separation, oligosaccharides were separated mainly based on the charge and the number of sugar residues.…”

Section: ·2 Graphitized Carbon Chromatographymentioning

confidence: 99%

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

Suzuki

2013

ANAL. SCI.

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“…2-Aminobenzoic acid (2-AA) is a useful derivatizing reagent for carbohydrate analyses. [11][12][13][14][15][16][17][18] The 2-AA labeling procedure is rapid, selective, and easy to perform. 14 Moreover, 2-AA derivatives are able to become charged positively, negatively, or neutral, depending on the pH of a running buffer, since it has both a carboxyl group and an amino group.…”

mentioning

confidence: 99%

Separation of 2-Aminobenzoic Acid-Derivatized Glycosaminoglycans and Asparagine-linked Glycans by Capillary Electrophoresis

Sato

Ōkubo

et al. 2005

ANAL. SCI.

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Glycosylation is known to be the most common and diverse post-translational modification of proteins. It may affect their biological activity, solubility, stability, and immunogenicity. Therefore, determining the sugar structures has become increasingly important to understand and elucidate the functions of these molecules.To analyze carbohydrates, a high separation efficiency is required, because oligosaccharides have branched structures and comprise a variety of isomers and homologues. Chromatographic methods have recently been commonly used to identify the oligosaccharide structures in a glycoprotein. They are powerful methods for the structural characterization of N-linked oligosaccharides, and consume only picomoles of samples. For example, a two-dimensional sugar mapping technique for pyridylaminated oligosaccharides 1-3 or a highperformance anion-exchange chromatography with a pulsed amperometric detector 4 are well known as high-resolution and sensitive methods.High-performance capillary electrophoresis (HPCE), which is a relatively new separation technique, is known as a highresolution, simple and rapid method. Usually, it does not require any pretreatment steps, and permits analyses by a variety of separation modes simply by changing the selection of a running electrolyte solution. Thus, HPCE has extended its applicability to various kinds of carbohydrates, including monosaccharides, oligosaccharides, glycosaminoglycans (GAGs), and glycans of glycoproteins.5-10 These methods are superior in small sample consumption and easy operations to other methods including HPLC analyses.Because most of carbohydrates do not have strong absorbance in UV and visible regions, labeling procedures are necessary for HPCE analyses. 2-Aminobenzoic acid (2-AA) is a useful derivatizing reagent for carbohydrate analyses. [11][12][13][14][15][16][17][18] The 2-AA labeling procedure is rapid, selective, and easy to perform. 14 Moreover, 2-AA derivatives are able to become charged positively, negatively, or neutral, depending on the pH of a running buffer, since it has both a carboxyl group and an amino group. 15 This charge flexibility is suitable for multidimensional HPCE analysis. We have reported on a highly efficient derivatizing method and the determination of 2-AA-derivatized monosaccharides by HPCE with UV detection. 15,17,18 This method has advantages of simplicity, rapidness, high resolution, and high sensitivity. In the present paper, we report that the HPCE separations of 2-AA-derivatized GAGs and N-linked glycans of glycoproteins have also been achieved. Experimental ReagentsAll of the reagents were of the highest grade available and used without any further purification. Sodium cyanoborohydride was from Aldrich. Various sulfated heparinderived-∆-disaccharides and heparan sulfate-derived-∆-disaccharides, chondroitin sulfate-∆-disaccharides, hyaluronic acid, keratan sulfate from bovine cornea, glucose-oligomer, Nglycan, chondroitinase ABC, and keratenase II (Bacillus sp.) were all purchased from Seikagaku Kogyo Corp....

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High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid

Cited by 261 publications

References 21 publications

Characterization of Alzheimer's β-Secretase Protein BACE

Characterization of Alzheimer's β-Secretase Protein BACE

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

Separation of 2-Aminobenzoic Acid-Derivatized Glycosaminoglycans and Asparagine-linked Glycans by Capillary Electrophoresis

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