197
11.8.1 Carbohydrate: BSA Ratio
MALDI-TOF-MS analysis of one of these synthetic vaccines allowed to observe the
following protonated molecular ions: [M + H]
+
at m/z 71,448.36 and [M + 2H]
2+
at
m/z 35,730.08. The molecular weight of this vaccine was thus found to be
71,447.36 Da. As the molecular weight of the synthetic tetrasaccharide side chain of
the collagen-like region being 950.43 Da, the carbohydrate: protein ratio of the
synthetic vaccine was determined to be 5.4:1.
11.8.2 Glycation Sites Determination
The determination of the glycation sites of the hapten-BSA vaccine neoglycoconjugate was carried out by enzymatic digestion with the trypsin and/or GluC V8 proteases, followed by the MALDI-MS/MS and LC-MS/MS analysis of the digests.
The enzymatic digestion of the hapten-BSA glycoconjugate and MALDI-MS/
MS, as well as LC-MS/MS analyses, were carried out as previously described
[67–71].
11.8.2.1 MALDI-TOF/TOF-MS/MS
The spectra obtained during the MALDI-TOF/TOF-MS /MS analysis of the tryptic
and GluC V8 digests (Fig. 11.7) were submitted to the MASCOT library to identify
by PMF the peptides matching to the BSA. Two Serum Albumin protein isoforms
from the Bos taurus species were identified for the tryptic and GluC V8 digests: the
serum albumin precursor (gi|1,351,907) and the serum albumin (gi|74,267,962).
The detected peptides that were not identified in the database were analyzed by
tandem mass spectrometry. The MALDI-MS/MS analysis of the tryptic digests
allowed to identify three glycated peptides corresponding to BSA peptides with an
increment of 950 Da to their original molecular mass, namely: ALK*AWSVAR
at m/z 1951.0130 (Fig. 11.8), VTK*CCTESLVNR at m/z 2416.1372, and
QNCDQFEK*LGEYGFQNALIVR at m/z 3478.6429 (glycation represented with
an asterisk on the lysine residue).
The high-energy CID-MS/MS analysis of the glycopeptide ALK*AWSVAR
(Fig. 11.8) at m/z 1951.0130 afforded a series of product ions corresponding to the
entire precursor ion that losses different carbohydrate portions, the monosaccharide
B 1 (−259 Da), disaccharide B 2 (−405 Da), trisaccharide B 3 (−551 Da) and/or the
tetrasaccharide B 4 (−697 Da), leading respectively to the formation of the following
productions: Y 3
+
at m/z 1691.8829, Y 2
+
at m/z 1545.8376, Y1
+
at m/z 1399.7768 and
Y0
+
at m/z 1253.7010 (Fig. 11.8). The mass difference between Y 3
+
and Y 2
+
, Y 2
+
and
Y 1
+
, Y 1
+,
and Y 0
+
were found to correspond to one α-L-rhamnopyranosyl unit
(146 Da). Moreover, product ions resulting from the fragmentation of the same
11 Defense Against Biological Terrorism: Vaccines and Their Characterizations
11.8.1 Carbohydrate: BSA Ratio
MALDI-TOF-MS analysis of one of these synthetic vaccines allowed to observe the
following protonated molecular ions: [M + H]
+
at m/z 71,448.36 and [M + 2H]
2+
at
m/z 35,730.08. The molecular weight of this vaccine was thus found to be
71,447.36 Da. As the molecular weight of the synthetic tetrasaccharide side chain of
the collagen-like region being 950.43 Da, the carbohydrate: protein ratio of the
synthetic vaccine was determined to be 5.4:1.
11.8.2 Glycation Sites Determination
The determination of the glycation sites of the hapten-BSA vaccine neoglycoconjugate was carried out by enzymatic digestion with the trypsin and/or GluC V8 proteases, followed by the MALDI-MS/MS and LC-MS/MS analysis of the digests.
The enzymatic digestion of the hapten-BSA glycoconjugate and MALDI-MS/
MS, as well as LC-MS/MS analyses, were carried out as previously described
[67–71].
11.8.2.1 MALDI-TOF/TOF-MS/MS
The spectra obtained during the MALDI-TOF/TOF-MS /MS analysis of the tryptic
and GluC V8 digests (Fig. 11.7) were submitted to the MASCOT library to identify
by PMF the peptides matching to the BSA. Two Serum Albumin protein isoforms
from the Bos taurus species were identified for the tryptic and GluC V8 digests: the
serum albumin precursor (gi|1,351,907) and the serum albumin (gi|74,267,962).
The detected peptides that were not identified in the database were analyzed by
tandem mass spectrometry. The MALDI-MS/MS analysis of the tryptic digests
allowed to identify three glycated peptides corresponding to BSA peptides with an
increment of 950 Da to their original molecular mass, namely: ALK*AWSVAR
at m/z 1951.0130 (Fig. 11.8), VTK*CCTESLVNR at m/z 2416.1372, and
QNCDQFEK*LGEYGFQNALIVR at m/z 3478.6429 (glycation represented with
an asterisk on the lysine residue).
The high-energy CID-MS/MS analysis of the glycopeptide ALK*AWSVAR
(Fig. 11.8) at m/z 1951.0130 afforded a series of product ions corresponding to the
entire precursor ion that losses different carbohydrate portions, the monosaccharide
B 1 (−259 Da), disaccharide B 2 (−405 Da), trisaccharide B 3 (−551 Da) and/or the
tetrasaccharide B 4 (−697 Da), leading respectively to the formation of the following
productions: Y 3
+
at m/z 1691.8829, Y 2
+
at m/z 1545.8376, Y1
+
at m/z 1399.7768 and
Y0
+
at m/z 1253.7010 (Fig. 11.8). The mass difference between Y 3
+
and Y 2
+
, Y 2
+
and
Y 1
+
, Y 1
+,
and Y 0
+
were found to correspond to one α-L-rhamnopyranosyl unit
(146 Da). Moreover, product ions resulting from the fragmentation of the same
11 Defense Against Biological Terrorism: Vaccines and Their Characterizations