6
Disulfide Bond Analysis on Orbitrap Mass Spectrometry
Conclusion
Two different digestion ways were used in this study: one employed trypsin only
while the other used the combination of chymotrypsin and trypsin.
10 out of 16 disulfide bonds were identified in trypsin-only digestion experiment
and 13 out of 16 were found in double digestion way, and the results were
complementary.
Totally, 15 out of 16 disulfide bonds were identified and all of them have confident
MS/MS information.
Biopharmaceutical industry-related disulfide bonds analysis were performed on
Orbitrap Elite, with reproducible and high confident mass information.
References
1. Nebija D., Kopelent-Frank H., Urban E., Noe C. R. and Lachmann, B.
Comparison of two-dimensional gel electrophoresis patterns and MALDI-TOF
MS analysis of therapeutic recombinant monoclonal antibodies trastuzumab and
rituximab.
Journal of Pharmaceutical and Biomedical Analysis, 2011, 56, 684
–
91.
2. Martin Samonig, Christian Huber and Kai Scheffler
. LC/MS Analysis of the
Monoclonal Antibody Rituximab Using the Q Exactive Benchtop Orbitrap Mass
Spectrometer.
3.
FIGURE 8. All of the identified disulfide linkage of Rituximab. Red, the disulfide
bond can be found in both experiments. Blue , the disulfide bond can just be
found in trypsin-only digestion. Green, Blue , the disulfide bond can be found
only in double digestion.
sulfide bond C133-C193 on light
FIGURE 7 shows the disulfide bond between Cys133 and Cys193 on light chain. In
this double digestion experiment, which employs chymotrypsin and trypsin at the same
time. Because the disulfide bond-included peptides are usually very large, to get more
shorter peptides, double digestion is a good choice.
In double digestion result, we can find that the intrachain linkage on the Fab region of
heavy chain and one of the interchain between heavy chains are identified, which
weren’t identified in
trypsin only experiment. It is worth noting that the C265-C325
linkage on heavy chain was found in trypsin only experiment, but not identified in
double digestion condition; this can be explain that the peptide which contains this
linkage was cut into some very short parts
——
only one or two amino acids, which was
beyond the detection line of mass spectrometer.
By combination of these two results, 15 out of 16 disulfide bonds on Rituximab were
identified in our experiment successfully.
linkage between Cys265 and
is high (2.19ppm), which
ccurate data. It’s also easily to
gment ions were identified.
ence of the disulfide bond.
oduce more disulfide linkage-
meter detection. Chymotrypsin
et nearly 100% sequence
disulfide bonds in the double
ll identified disulfide bonds in
ouble digested Rituximab.
Rituxin is a registered trademark of Biogen Idec Inc. MabThera is a registered trademark of F. Hoffman-LaRoche
Ltd.
StavroX is a trademark of Martin-Luther University of Halle-Wittenberg, Germany. All other trademarks are the
property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the
intellectual property rights of others.
PO64155-EN 0614S
