192
11.7.4 Molecular Weight and Carbohydrate-to-Protein
Ratio Determination
The aim of determining the molecular weight and the carbohydrate-to-protein ratio
of a carbohydrate-protein neoglycoconjugate is to define the number of carbohydrates that are incorporated in the protein carrier, as a result of the conjugation. Two
main methods are currently used for the molecular weight determination
carbohydrate- protein glycoconjugates: matrix-assisted-laser-ionization time-offlight mass spectrometry (MALDI-TOF-MS) [56, 57] and surface-enhanced laser
desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) [53–55,
58–60]. Both of these methods allow determining the carbohydrate-to-protein ratio
of the neoglycoconjugates by comparing the molecular weight of the protein before
and after the conjugation to that of the neoglycoconjugate.
11.7.5 Mass Spectrometry Methods for the Characterization
of Carbohydrate Vaccines
Mass spectrometry has emerged as a powerful technique for the characterization of
different biomolecules ranging from small molecules to larger molecules. Thus,
mass spectrometry is extensively used in proteomics [61], glycomics [62], metabolomics [63], lipidomics [64], and in oligonucleotides [65] analysis.
Initially, the exploring of MALDI-TOF-MS of the different hapten-BSA glycoconjugate vaccines allowed us to determine the hapten-to-BSA ratios. Then the glycoconjugate vaccine samples were then digested and analyzed by MALDI-TOF/
TOF-MS/MS and LC-ESI-QqTOF-MS/MS for the determination of glycation sites.
The digestion was done by two different enzymes; trypsin, which will not be able
to digest or react with the glycated lysines of the protein, and the other enzyme was
GluC V8 endoproteinase which is known to digest proteins at C-terminus of the
aspartic acid and glutamic acid residues. Finally, the MS/MS spectra will be submitted to the MASCOT library to get the matched and non-matched peptides.
11.7.6 MALDI-TOF-MS
Matrix-assisted laser desorption ionization mass spectrometry has been successfully used for the determination of the molecular weight of biomolecules, such as
proteins, oligosaccharides, and glycoproteins [56–58]. Kamath et al. used MALDITOF- MS to characterize neoglycoconjugates formed by the conjugation of oligosaccharide amines to carrier proteins by the aim of diethyl squarate [59]. The
MALDI-MS was recorded in linear mode and with positive ion detection. Figure 11.4
displays the MALDI-TOF-MS analysis of BSA (A) and the following
M. Bologna et al.
11.7.4 Molecular Weight and Carbohydrate-to-Protein
Ratio Determination
The aim of determining the molecular weight and the carbohydrate-to-protein ratio
of a carbohydrate-protein neoglycoconjugate is to define the number of carbohydrates that are incorporated in the protein carrier, as a result of the conjugation. Two
main methods are currently used for the molecular weight determination
carbohydrate- protein glycoconjugates: matrix-assisted-laser-ionization time-offlight mass spectrometry (MALDI-TOF-MS) [56, 57] and surface-enhanced laser
desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) [53–55,
58–60]. Both of these methods allow determining the carbohydrate-to-protein ratio
of the neoglycoconjugates by comparing the molecular weight of the protein before
and after the conjugation to that of the neoglycoconjugate.
11.7.5 Mass Spectrometry Methods for the Characterization
of Carbohydrate Vaccines
Mass spectrometry has emerged as a powerful technique for the characterization of
different biomolecules ranging from small molecules to larger molecules. Thus,
mass spectrometry is extensively used in proteomics [61], glycomics [62], metabolomics [63], lipidomics [64], and in oligonucleotides [65] analysis.
Initially, the exploring of MALDI-TOF-MS of the different hapten-BSA glycoconjugate vaccines allowed us to determine the hapten-to-BSA ratios. Then the glycoconjugate vaccine samples were then digested and analyzed by MALDI-TOF/
TOF-MS/MS and LC-ESI-QqTOF-MS/MS for the determination of glycation sites.
The digestion was done by two different enzymes; trypsin, which will not be able
to digest or react with the glycated lysines of the protein, and the other enzyme was
GluC V8 endoproteinase which is known to digest proteins at C-terminus of the
aspartic acid and glutamic acid residues. Finally, the MS/MS spectra will be submitted to the MASCOT library to get the matched and non-matched peptides.
11.7.6 MALDI-TOF-MS
Matrix-assisted laser desorption ionization mass spectrometry has been successfully used for the determination of the molecular weight of biomolecules, such as
proteins, oligosaccharides, and glycoproteins [56–58]. Kamath et al. used MALDITOF- MS to characterize neoglycoconjugates formed by the conjugation of oligosaccharide amines to carrier proteins by the aim of diethyl squarate [59]. The
MALDI-MS was recorded in linear mode and with positive ion detection. Figure 11.4
displays the MALDI-TOF-MS analysis of BSA (A) and the following
M. Bologna et al.