194
11.7.7 SELDI-TOF-MS
Surface-enhanced laser desorption ionization time-of-flight mass spectrometry
(SELDI-TOF-MS) has been extensively used for biomarkers discovery [59], and
cancer diagnosis [60]. This technique consists in using a modified target for the
immunoaffinity purification of proteins before analysis.
The group of Kováč successfully used this analytical method to monitor the conjugation of synthetic carbohydrate antigens to different protein carriers [53–55, 66].
Figure 11.5 shows the SELDI-TOF-MS analysis with a ProteinChip® System of a
neoglycoconjugate prepared by the dialkyl squarate chemistry attachment of the
hexasaccharide of Vibrio cholerae O:1 to the BSA [65]. This technology allowed
the analysis of neoglycoconjugates at different reaction times by taking an aliquot
of the reaction mixture, in the picomolar concentration range, and analyzing it without any purification step. In addition, this method was found to be fast, and only a
small amount of sample is used for analysis. The same group followed the progress
of conjugation of the hexasaccharide (exact mass: 1780.79 Da) to the BSA protein
(molecular mass: 66,430 Da) at different reaction times [65]. It was observed that
the conjugation rate increased with the reaction time. In addition, the SELDITOF- MS also allowed observing that after a reaction time of 7 h, the fine structure
the peak of the neoglycoconjugate (Fig. 11.6) shows the polydispersity of the neoglycoconjugate formed from the hexasaccharide and BSA.
Moreover, after the conjugation is complete, the excess oligosaccharide can be
recovered for further use, allowing an economy of labor and time for the preparation
of synthetic oligosaccharides and their conjugation [66].
11.7.8 Glycation Sites Determination
The glycation site’s determination of carbohydrates-protein neoglycoconjugates is
usually carried out by first digesting the neoglycoconjugate using a protease, such
as trypsin or GluC V8 endoproteinase, followed by MALDI-TOF-MS/MS or liquid
chromatography-tandem mass spectrometry [67–72].
It has to be noted that during the tandem mass spectrometry analysis of the glycoconjugate digests, the identification of the glycated peptides is confirmed by the
presence of diagnostic product ions of the carbohydrate in the mass spectrum.
In addition, the tandem mass spectrometry analysis also reveals the sequence of
the peptide through diagnostic product ions of the peptide moiety of the glycated
peptide. The combined information allows the unambiguous characterization of the
carbohydrate-peptide and the glycation site identification. It has to be noted that
during the tandem mass spectrometry analyses of the glycated peptides, the product
ions corresponding to the fragmentation of the peptide portion were identified using
the nomenclature established by Roepstorff et al. and lately modified by Johnson
and coworkers [73, 74], and the product ions resulting from the fragmentation of the
M. Bologna et al.
11.7.7 SELDI-TOF-MS
Surface-enhanced laser desorption ionization time-of-flight mass spectrometry
(SELDI-TOF-MS) has been extensively used for biomarkers discovery [59], and
cancer diagnosis [60]. This technique consists in using a modified target for the
immunoaffinity purification of proteins before analysis.
The group of Kováč successfully used this analytical method to monitor the conjugation of synthetic carbohydrate antigens to different protein carriers [53–55, 66].
Figure 11.5 shows the SELDI-TOF-MS analysis with a ProteinChip® System of a
neoglycoconjugate prepared by the dialkyl squarate chemistry attachment of the
hexasaccharide of Vibrio cholerae O:1 to the BSA [65]. This technology allowed
the analysis of neoglycoconjugates at different reaction times by taking an aliquot
of the reaction mixture, in the picomolar concentration range, and analyzing it without any purification step. In addition, this method was found to be fast, and only a
small amount of sample is used for analysis. The same group followed the progress
of conjugation of the hexasaccharide (exact mass: 1780.79 Da) to the BSA protein
(molecular mass: 66,430 Da) at different reaction times [65]. It was observed that
the conjugation rate increased with the reaction time. In addition, the SELDITOF- MS also allowed observing that after a reaction time of 7 h, the fine structure
the peak of the neoglycoconjugate (Fig. 11.6) shows the polydispersity of the neoglycoconjugate formed from the hexasaccharide and BSA.
Moreover, after the conjugation is complete, the excess oligosaccharide can be
recovered for further use, allowing an economy of labor and time for the preparation
of synthetic oligosaccharides and their conjugation [66].
11.7.8 Glycation Sites Determination
The glycation site’s determination of carbohydrates-protein neoglycoconjugates is
usually carried out by first digesting the neoglycoconjugate using a protease, such
as trypsin or GluC V8 endoproteinase, followed by MALDI-TOF-MS/MS or liquid
chromatography-tandem mass spectrometry [67–72].
It has to be noted that during the tandem mass spectrometry analysis of the glycoconjugate digests, the identification of the glycated peptides is confirmed by the
presence of diagnostic product ions of the carbohydrate in the mass spectrum.
In addition, the tandem mass spectrometry analysis also reveals the sequence of
the peptide through diagnostic product ions of the peptide moiety of the glycated
peptide. The combined information allows the unambiguous characterization of the
carbohydrate-peptide and the glycation site identification. It has to be noted that
during the tandem mass spectrometry analyses of the glycated peptides, the product
ions corresponding to the fragmentation of the peptide portion were identified using
the nomenclature established by Roepstorff et al. and lately modified by Johnson
and coworkers [73, 74], and the product ions resulting from the fragmentation of the
M. Bologna et al.