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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 linear gluco-tetrasaccharides with mixed linkages and branched gluco-decasaccharides. (A) Pullu-4 (α1,6,1,4,1,4 linkages). (B) Barley-4c (β1,4,1,3,1,4 linkages). (C) Barley-4-b (β1,4, 1,4, 1,3 linkages). (D) HE-10B2. (E) HE-10B3. (F) HE-10B5.
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Figure 3: Negative-ion ESI-CID-MS/MS product-ion spectra of linear gluco-tetrasaccharides with mixed linkages and branched gluco-decasaccharides. (A) Pullu-4 (α1,6,1,4,1,4 linkages). (B) Barley-4c (β1,4,1,3,1,4 linkages). (C) Barley-4-b (β1,4, 1,4, 1,3 linkages). (D) HE-10B2. (E) HE-10B3. (F) HE-10B5.

Mentions: The principles established above were used to assess the utility of the method for sequencing linear gluco-oligosaccharides containing different linkage types. Pullu-4 has two1,4-linkages and one 1,6-linkage (supplemental Table S3). The product-ion spectrum (Fig. 3A) can be used to assign the three individual linkages as well as their locations. The two A-ion sets derived from losses of 60/78/120 recorded from the two consecutive residues at the reducing side were identical to the fragmentation pattern for the 4-linked Glc (Table IB), corresponding to two 1,4-linkages. The ion set with -60/90/120 from C2 (m/z 341) (Table IB) can be used to assign the nonreducing terminal 1,6-linkage. Other α1,4/α1,6-linked gluco-oligosaccharides Pullu-7 (supplemental Fig. S3) and the isomeric trisaccharide pair Pano-3 and i-Pano-3 (supplemental Fig. S4) can also be readily identified.


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 linear gluco-tetrasaccharides with mixed linkages and branched gluco-decasaccharides. (A) Pullu-4 (α1,6,1,4,1,4 linkages). (B) Barley-4c (β1,4,1,3,1,4 linkages). (C) Barley-4-b (β1,4, 1,4, 1,3 linkages). (D) HE-10B2. (E) HE-10B3. (F) HE-10B5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Negative-ion ESI-CID-MS/MS product-ion spectra of linear gluco-tetrasaccharides with mixed linkages and branched gluco-decasaccharides. (A) Pullu-4 (α1,6,1,4,1,4 linkages). (B) Barley-4c (β1,4,1,3,1,4 linkages). (C) Barley-4-b (β1,4, 1,4, 1,3 linkages). (D) HE-10B2. (E) HE-10B3. (F) HE-10B5.
Mentions: The principles established above were used to assess the utility of the method for sequencing linear gluco-oligosaccharides containing different linkage types. Pullu-4 has two1,4-linkages and one 1,6-linkage (supplemental Table S3). The product-ion spectrum (Fig. 3A) can be used to assign the three individual linkages as well as their locations. The two A-ion sets derived from losses of 60/78/120 recorded from the two consecutive residues at the reducing side were identical to the fragmentation pattern for the 4-linked Glc (Table IB), corresponding to two 1,4-linkages. The ion set with -60/90/120 from C2 (m/z 341) (Table IB) can be used to assign the nonreducing terminal 1,6-linkage. Other α1,4/α1,6-linked gluco-oligosaccharides Pullu-7 (supplemental Fig. S3) and the isomeric trisaccharide pair Pano-3 and i-Pano-3 (supplemental Fig. S4) can also be readily identified.

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