6
Middle-down Analysis of Monoclonal Antibody Middle using Nano-flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass Spectrometer
b
by LC-MS analysis of the
of light chain,
m/z
1117.87 of Fd’ and
ed respectively for ETD and HCD
FIGURE 9. Comparison of experimental C
57
ion of Fc/2+G0F chain to
theoretically predicted isotope distribution. This ion is critical for identifying
and localizing G0F on Asn61 residue.
719.3735
719 2621
s was applied for ETD
D was 15-25 %. As shown in Figure
ultiply charged fragment ions.
sequence was performed using
CD suggest 50% sequence
60
80
100
Abundance
z=9
.
z=9
719.4851
z=9
719.1510
z 9
719.5956
C
57
, Experimental
<3ppm
G0F and 32% coverage for Fd’
n of pyroglutamate of Fd’chain was
d HCD. Both intact and tandem
ly fragmenting the peptide back bone
0
20
40
Relative
=
z=9
719.0392
z=9
719.7081
z=9
719.8182
z=9
719 2635719 3749
been recognized as the method of
ding glycosylation. Multiple identified
ously located the glycan G0F on
pectrum, the high resolution and
dent identification of low abundance
60
80
100
.
z=9
.
z=9
719.4863
z=9
719.1521
z=9
719.5977
z=9
719.0407
C
57
, Theoretical
terference. For example, shown in
C
61
ion with G0F; Figure 9 presents
though not all the isotopic peaks of
nce, all seven isotopes identified
tion). Both ETD ions, C
61
and C
57,
0
20
40
z=9
719.7090
z=9
719.8204
z=9
718.9293
z=9
tion of G0F on Asn61 residue.
FIGURE 10. ETD (blue) and HCD (red) coverage of light chain.
ecursor ion as
m/z
902.20,
spectrum covering C61 ion at
rms the addition of G0F
719.0
719.2
719.4
719.6
719.8
60
70
80
90
100
undance
939.2334
z=9
939.1216
z=9
939.3439
z=9
939.5667
z=9
+9, C
61
with
G0F, <3ppm
0
10
20
30
40
50
Relative Ab
FIGURE 11. ETD (blue) and HCD (red) coverage of Fd’ chain.
939.0 939.2 939.4 939.6
m/z
7858
3
1203.0937
z=4
1073 0261 .
z=6
1528.8462
z=1
1287.6293
z=5
1000
1200
1400
Conclusion
The middle-down study indicated that complementary fragmentation mechanisms allow
Fc/2+G0F chain. Asn61 was
extensive sequence coverage as well as identification and localization of PTMs
including labile glycosylation. The middle-down approach produced detailed structure
information deep into the middle of the mAb chains especially in the epitope region.
Acknowledgements
We would like to thank National Institute of Standards and Technology for providing the
intact mAb protein sample (candidate NIST RM 8670 mAb lot #3F1b).
FabRICATOR® is a registered trademark for recombinant IdeS. 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
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PO64147-EN 0614S
