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Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.

Palma AS, Liu Y, Zhang H, Zhang Y, McCleary BV, Yu G, Huang Q, Guidolin LS, Ciocchini AE, Torosantucci A, Wang D, Carvalho AL, Fontes CM, Mulloy B, Childs RA, Feizi T, Chai W - Mol. Cell Proteomics (2015)

Bottom Line: The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans.The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN.The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom; §UCIBIO-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, NOVA University of Lisbon; angelina.palma@fct.unl.pt w.chai@imperial.ac.uk.

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Negative-ion ESI-CID-MS/MS product-ion spectra of gluco-disaccharides and linear gluco-heptasaccharides with homo-linkages. (A) Kojibiose with an α1,2-linkage. (B) Nigerose with an α1,3-linkage. (C) Maltobiose (Malto-2) with an α1,4-linkage. (D) Isomaltobiose with an α1,6-linkage. (E) Cyano-7 with α1,2-linkages. (F) Lam-7 with β1,3-linkages. (G) Malto-7 with α1,4-linkages. (H) Dext-7 with α1,6- linkages.
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Figure 2: Negative-ion ESI-CID-MS/MS product-ion spectra of gluco-disaccharides and linear gluco-heptasaccharides with homo-linkages. (A) Kojibiose with an α1,2-linkage. (B) Nigerose with an α1,3-linkage. (C) Maltobiose (Malto-2) with an α1,4-linkage. (D) Isomaltobiose with an α1,6-linkage. (E) Cyano-7 with α1,2-linkages. (F) Lam-7 with β1,3-linkages. (G) Malto-7 with α1,4-linkages. (H) Dext-7 with α1,6- linkages.

Mentions: The product-ion spectra of the four α-anomeric disaccharides with 1,2-, 1,3-, 1,4-, and 1,6-linkages are shown in Fig. 2A-2D. Under the conditions selected each glucose linkage isomer had a different fragmentation pattern. Glycosidic C-type (43) cleavages resulting in fragment ions C1 at m/z 179, arising from a neutral loss of 162 Da from the molecular ion at m/z 341, were observed in the spectra of all disaccharides but with different relative intensities. A-type fragmentation across the saccharide ring gave particularly useful information on the linkages: In the spectrum of the α1,2-linked kojibiose (Fig. 2A), neutral losses of 18, 78 and 120 Da gave a distinctive set of three ions at m/z 323, 263 and 221, as [M-H]−-h, 0,4A2-h, and 0,2A2, respectively (-h denotes dehydration). For the α1,3-linked nigerose, glycosidic cleavage C1 only was observed at m/z 179, whereas A-type fragmentation was absent (Fig. 2B). The α1,4- and α1,6-linked disaccharides each produced extensive A-type fragmentation in addition to the C1 ions: in the spectrum of the α1,4-linked maltobiose (Fig. 2C) these were the 0,2A2 (m/z 281), 0,2A2-h (m/z 263), and 2,4A2 (m/z 221) arising from neutral losses of 60, 78 and 120 Da, respectively, whereas in the spectrum of α1,6-linked isomaltobiose (Fig. 2D), these were the 0,2A2 (m/z 281), 0,3A2 (m/z 251), and 0,4A2 (m/z 221) arising from neutral losses of 60, 90, and 120 Da, respectively. The β-anomers of the 1,2-, 1,3-, 1,4-, and 1,6-linked disaccharides (Table IA) gave product-ion spectra (not shown) almost identical to those of their α-anomers.


Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.

Palma AS, Liu Y, Zhang H, Zhang Y, McCleary BV, Yu G, Huang Q, Guidolin LS, Ciocchini AE, Torosantucci A, Wang D, Carvalho AL, Fontes CM, Mulloy B, Childs RA, Feizi T, Chai W - Mol. Cell Proteomics (2015)

Negative-ion ESI-CID-MS/MS product-ion spectra of gluco-disaccharides and linear gluco-heptasaccharides with homo-linkages. (A) Kojibiose with an α1,2-linkage. (B) Nigerose with an α1,3-linkage. (C) Maltobiose (Malto-2) with an α1,4-linkage. (D) Isomaltobiose with an α1,6-linkage. (E) Cyano-7 with α1,2-linkages. (F) Lam-7 with β1,3-linkages. (G) Malto-7 with α1,4-linkages. (H) Dext-7 with α1,6- linkages.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4390274&req=5

Figure 2: Negative-ion ESI-CID-MS/MS product-ion spectra of gluco-disaccharides and linear gluco-heptasaccharides with homo-linkages. (A) Kojibiose with an α1,2-linkage. (B) Nigerose with an α1,3-linkage. (C) Maltobiose (Malto-2) with an α1,4-linkage. (D) Isomaltobiose with an α1,6-linkage. (E) Cyano-7 with α1,2-linkages. (F) Lam-7 with β1,3-linkages. (G) Malto-7 with α1,4-linkages. (H) Dext-7 with α1,6- linkages.
Mentions: The product-ion spectra of the four α-anomeric disaccharides with 1,2-, 1,3-, 1,4-, and 1,6-linkages are shown in Fig. 2A-2D. Under the conditions selected each glucose linkage isomer had a different fragmentation pattern. Glycosidic C-type (43) cleavages resulting in fragment ions C1 at m/z 179, arising from a neutral loss of 162 Da from the molecular ion at m/z 341, were observed in the spectra of all disaccharides but with different relative intensities. A-type fragmentation across the saccharide ring gave particularly useful information on the linkages: In the spectrum of the α1,2-linked kojibiose (Fig. 2A), neutral losses of 18, 78 and 120 Da gave a distinctive set of three ions at m/z 323, 263 and 221, as [M-H]−-h, 0,4A2-h, and 0,2A2, respectively (-h denotes dehydration). For the α1,3-linked nigerose, glycosidic cleavage C1 only was observed at m/z 179, whereas A-type fragmentation was absent (Fig. 2B). The α1,4- and α1,6-linked disaccharides each produced extensive A-type fragmentation in addition to the C1 ions: in the spectrum of the α1,4-linked maltobiose (Fig. 2C) these were the 0,2A2 (m/z 281), 0,2A2-h (m/z 263), and 2,4A2 (m/z 221) arising from neutral losses of 60, 78 and 120 Da, respectively, whereas in the spectrum of α1,6-linked isomaltobiose (Fig. 2D), these were the 0,2A2 (m/z 281), 0,3A2 (m/z 251), and 0,4A2 (m/z 221) arising from neutral losses of 60, 90, and 120 Da, respectively. The β-anomers of the 1,2-, 1,3-, 1,4-, and 1,6-linked disaccharides (Table IA) gave product-ion spectra (not shown) almost identical to those of their α-anomers.

Bottom Line: The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans.The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN.The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Glycosciences Laboratory, Department of Medicine, Imperial College London, United Kingdom; §UCIBIO-REQUIMTE, Department of Chemistry, Faculty of Science and Technology, NOVA University of Lisbon; angelina.palma@fct.unl.pt w.chai@imperial.ac.uk.

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Related in: MedlinePlus