200
establishing a diagnostic fragmentation signature of the synthetic tetrasaccharide. In
addition, the fragmentation of the peptide portion led to the formation of b- and yproduct ions that allowed the determination of the sequence of the glycated peptide.
However, some peptide product ions had the particularity to have lost the carbohydrate portion but were still attached to the spacer-squaric acid chain: [b 3 – B 4 ]
+
at
m/z 565.2661, [b 4 – B 4 ]
+
at m/z 636.2715 and [y 7 –B 2 ]
+
at m/z 1361.7265. Thus, the
identified glycated peptides ALK*AWSVAR at m/z 1951.0130, VTK*CCTESLVNR
at m/z 2416.1372, and QNCDQFEK*LGEYGFQNALIVR at m/z 3478.6429
allowed to determine the glycation site on the following lysine residues: Lys 235,
Lys 498 and Lys 420, respectively.
Similarly, the MALDI-MS/MS analysis of the GluC V8 digests afforded different glycated peptides: LCK*VASLRE at m/z 2025.0186, YAVSVLLRLAK*E at
m/z 2311.2476 and YAVSVLLRLAK*EYE at m/z 2603.3484, allowing to identify
the following glycation sites on lysine residues: Lys 100 and Lys 374. To sum it up,
only five glycation sites were identified during the MALDI-MS and MS/MS analyses of the tryptic and GluC V8 digests of the hapten-BSA glycoconjugate: Lys 100,
Lys 235, Lys 374, Lys 420 and Lys 498.
11.8.2.2 LC-MS/MS
The second approach for the determination of the glycation sites of the vaccine
neoglycoconjugate was the LC-MS/MS analysis of the tryptic and GluC V8 digests.
It was noted that the LC-MS/MS analysis of peptides has the advantage of minimizing the ionization suppression effect comparing to the MALDI-MS/MS analysis
[88, 89].
The data of the LC-MS/MS analysis of the tryptic and GluC V8 digests were
submitted to the MASCOT library and matched two serum albumin protein isoforms: the serum albumin precursor (gi|1,351,907) and serum albumin
(gi|74,267,962) from Bos taurus.
For the tryptic digests, the BSA sequence coverage was found to be 57% for the
serum albumin precursor from Bos taurus (gi|1,351,907) and 58% for serum albumin protein from Bos taurus (gi|74,267,962). The LC-MS/MS analysis of the hapten- BSA tryptic digests allowed the identification of 18 glycated peptides, reported
in Table 11.2.
The low-energy CID-MS/MS analysis of the extracted precursor ions of the glycated peptides allowed to localize the glycation sites on the following 18 lysine residues: Lys 140, Lys 155, Lys 156, Lys 204, Lys 211, Lys 228, Lys 235, Lys 304, Lys
374, Lys 401, Lys 420, Lys 437, Lys 455, Lys 463, Lys 495, Lys 498, Lys 547 and
Lys 559.
The LC-MS/MS analysis of the hapten-BSA vaccine GluC V8 digest allowed the
identification of the serum albumin from Bos taurus (gi|74,267,962) with a sequence
coverage of 42% and the precursor serum albumin from Bos taurus (gi|1,351,907)
with a sequence coverage of 45%, in the MASCOT database. Table 11.3 displays
the identified glycated peptides during the LC-MS/MS analysis of the GluC V8
M. Bologna et al.
establishing a diagnostic fragmentation signature of the synthetic tetrasaccharide. In
addition, the fragmentation of the peptide portion led to the formation of b- and yproduct ions that allowed the determination of the sequence of the glycated peptide.
However, some peptide product ions had the particularity to have lost the carbohydrate portion but were still attached to the spacer-squaric acid chain: [b 3 – B 4 ]
+
at
m/z 565.2661, [b 4 – B 4 ]
+
at m/z 636.2715 and [y 7 –B 2 ]
+
at m/z 1361.7265. Thus, the
identified glycated peptides ALK*AWSVAR at m/z 1951.0130, VTK*CCTESLVNR
at m/z 2416.1372, and QNCDQFEK*LGEYGFQNALIVR at m/z 3478.6429
allowed to determine the glycation site on the following lysine residues: Lys 235,
Lys 498 and Lys 420, respectively.
Similarly, the MALDI-MS/MS analysis of the GluC V8 digests afforded different glycated peptides: LCK*VASLRE at m/z 2025.0186, YAVSVLLRLAK*E at
m/z 2311.2476 and YAVSVLLRLAK*EYE at m/z 2603.3484, allowing to identify
the following glycation sites on lysine residues: Lys 100 and Lys 374. To sum it up,
only five glycation sites were identified during the MALDI-MS and MS/MS analyses of the tryptic and GluC V8 digests of the hapten-BSA glycoconjugate: Lys 100,
Lys 235, Lys 374, Lys 420 and Lys 498.
11.8.2.2 LC-MS/MS
The second approach for the determination of the glycation sites of the vaccine
neoglycoconjugate was the LC-MS/MS analysis of the tryptic and GluC V8 digests.
It was noted that the LC-MS/MS analysis of peptides has the advantage of minimizing the ionization suppression effect comparing to the MALDI-MS/MS analysis
[88, 89].
The data of the LC-MS/MS analysis of the tryptic and GluC V8 digests were
submitted to the MASCOT library and matched two serum albumin protein isoforms: the serum albumin precursor (gi|1,351,907) and serum albumin
(gi|74,267,962) from Bos taurus.
For the tryptic digests, the BSA sequence coverage was found to be 57% for the
serum albumin precursor from Bos taurus (gi|1,351,907) and 58% for serum albumin protein from Bos taurus (gi|74,267,962). The LC-MS/MS analysis of the hapten- BSA tryptic digests allowed the identification of 18 glycated peptides, reported
in Table 11.2.
The low-energy CID-MS/MS analysis of the extracted precursor ions of the glycated peptides allowed to localize the glycation sites on the following 18 lysine residues: Lys 140, Lys 155, Lys 156, Lys 204, Lys 211, Lys 228, Lys 235, Lys 304, Lys
374, Lys 401, Lys 420, Lys 437, Lys 455, Lys 463, Lys 495, Lys 498, Lys 547 and
Lys 559.
The LC-MS/MS analysis of the hapten-BSA vaccine GluC V8 digest allowed the
identification of the serum albumin from Bos taurus (gi|74,267,962) with a sequence
coverage of 42% and the precursor serum albumin from Bos taurus (gi|1,351,907)
with a sequence coverage of 45%, in the MASCOT database. Table 11.3 displays
the identified glycated peptides during the LC-MS/MS analysis of the GluC V8
M. Bologna et al.