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Jahouh F, Saksena R, Aiello D, Napoli A, Sindona G, Kovác P, Banoub JH
Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O-PS of Vibrio cholerae O1, serotype Ogawa, and BSA revealed by matrix-assisted laser desorption-ionization tandem mass spectrometry
Journal of Mass Spectrometry 45(10) (2010)
1148-1159
Vibrio cholerae O1 Ogawa
(Ancestor NCBI TaxID 127906,
species name lookup)
Taxonomic group: bacteria / Proteobacteria
(Phylum: Proteobacteria)
Host organism: Homo sapiens
Associated disease: diarrhea [ICD11:
ME05.1 
, ICD11:
SA55 ];
cholera [ICD11:
1A00 , ICD11:
XN7N1 ]
NCBI PubMed ID: 20860010Journal NLM ID: 9504818Publisher: Chichester, UK: Wiley
Correspondence: banoubjo
dfo-mpo.gc.ca
Institutions: Department of Chemistry, Memorial University of Newfoundland, Saint John's, NL, Canada
We present the MALDI-TOF/TOF-MS analyses of various hapten-bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer-equipped, terminal monosaccharide of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten-BSA neoglycoconjugates with different hapten-BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI-TOF/TOF-MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI-TOF/TOF-MS/MS of the glycated peptides. The product-ion scans of the protonated molecules were carried out with a MALDI-TOF/TOF-MS/MS tandem mass spectrometer equipped with a high-collision energy cell. The high-energy collision-induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y-series product ions was very useful for the sequencing of various peptides. The series of a- and b-product ions confirmed the sequence of the conjugated peptides.
hapten, Vibrio cholerae O1, BSA, neoglycoconjugate vaccine, MALDI-TOF/TOF-MS, MALDI-CID-TOF-TOF-MS/MS, protein carrier
Structure type: polymer chemical repeating unit
Location inside paper: p.1149, fig.1a
Aglycon: spacer-squaric acid-BSA
Trivial name: Ogawa neoglycoconjugate
Compound class: O-polysaccharide
Methods: conjugation, MALDI-TOF/TOF MS, MALDI-CID-TOF/TOF MS/MS, SELDI-TOF MS
Enzymes that release or process the structure: tryptic digestion
Comments, role: part of structure (see RR: 25670); Vibrio cholerae O1 serotype Ogawa
Related record ID(s): 25670
NCBI Taxonomy refs (TaxIDs): 127906
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Jahouh F, Saksena R, Aiello D, Napoli A, Sindona G, Kovác P, Banoub JH
Glycation sites in neoglycoglycoconjugates from the terminal monosaccharide antigen of the O-PS of Vibrio cholerae O1, serotype Ogawa, and BSA revealed by matrix-assisted laser desorption-ionization tandem mass spectrometry
Journal of Mass Spectrometry 45(10) (2010)
1148-1159
Vibrio cholerae O1 Ogawa
(Ancestor NCBI TaxID 127906,
species name lookup)
Taxonomic group: bacteria / Proteobacteria
(Phylum: Proteobacteria)
Host organism: Homo sapiens
Associated disease: diarrhea [ICD11:
ME05.1 , ICD11:
SA55 ];
cholera [ICD11:
1A00 , ICD11:
XN7N1 ]
NCBI PubMed ID: 20860010Journal NLM ID: 9504818Publisher: Chichester, UK: Wiley
Correspondence: banoubjo
dfo-mpo.gc.ca
Institutions: Department of Chemistry, Memorial University of Newfoundland, Saint John's, NL, Canada
We present the MALDI-TOF/TOF-MS analyses of various hapten-bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer-equipped, terminal monosaccharide of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten-BSA neoglycoconjugates with different hapten-BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI-TOF/TOF-MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI-TOF/TOF-MS/MS of the glycated peptides. The product-ion scans of the protonated molecules were carried out with a MALDI-TOF/TOF-MS/MS tandem mass spectrometer equipped with a high-collision energy cell. The high-energy collision-induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y-series product ions was very useful for the sequencing of various peptides. The series of a- and b-product ions confirmed the sequence of the conjugated peptides.
hapten, Vibrio cholerae O1, BSA, neoglycoconjugate vaccine, MALDI-TOF/TOF-MS, MALDI-CID-TOF-TOF-MS/MS, protein carrier
Structure type: monomer
Location inside paper: p.1149, fig.1a, p.1157, fig.4
Aglycon: spacer-squaric acid
Trivial name: fragment of O-polysaccharide
Methods: conjugation, MALDI-TOF/TOF MS, MALDI-CID-TOF/TOF MS/MS, SELDI-TOF MS
Enzymes that release or process the structure: tryptic digestion
Comments, role: terminal residue of O-antigen part; of structure (see RR: 25289); Vibrio cholerae O1 serotype Ogawa
Related record ID(s): 25289
NCBI Taxonomy refs (TaxIDs): 127906
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Jahouh F, Hou SJ, Kovác P, Banoub JH
Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry
Journal of Mass Spectrometry 46(10) (2011)
993-1003
|
3HOBut3Me-(1-4)-b-D-Quip4N2Me-(1-3)-a-L-Rhap-(1-3)-a-L-Rhap-(1-2)-a-L-Rhap-(1--/spacer-BSA/ |
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Bacillus anthracis
(NCBI TaxID 1392,
species name lookup)
Taxonomic group: bacteria / Firmicutes
(Phylum: Firmicutes)
Associated disease: infection due to Bacillus anthracis [ICD11:
XN94F ]
NCBI PubMed ID: 22012665Publication DOI: 10.1002/jms.1980Journal NLM ID: 9504818Publisher: Chichester, UK: Wiley
Correspondence: banoubjo
dfo-mpo.gc.ca
Institutions: Department of Chemistry, Memorial University of Newfoundland, Saint John's, NL, Canada
We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate-protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate-spacer-carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate.
neoglycoconjugate, proteinases, glycated peptides, MALDI-TOF/TOF-CID-MS/MS, LC-ESI-QqTOF-CID-MS/MS
Structure type: oligomer
Location inside paper: p.994, fig.1
Aglycon: spacer-BSA
Contained glycoepitopes: IEDB_133754,IEDB_136105,IEDB_225177,IEDB_885823
Methods: MALDI-TOF/TOF MS, MALDI-CID-TOF/TOF MS/MS, LC-ESI-QTOF MS/MS
Comments, role: synthetic tetrasaccharide
NCBI Taxonomy refs (TaxIDs): 1392
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There is only one chemically distinct structure:
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