6
Top-Down Analysis of Intact Antibodies Using Orbitrap Mass Spectrometry
FIGURE 8. EThcD mass spectrum of the intact Herceptin antibody in denatured
form.
100
1928.53125
R=104200
z=5
acquire HCD spectra of the
trometer at three different
t
m/z
400) to check if ultrahigh
pping isotopic clusters. An
er for the top-down analysis of
70
80
90
2052.18481
R=99700
z=5
1802.38403
R=106200
z=6
2162.85938
R=96300
z=5
414.19843
R=225401
z=1
263 fragment ions
(97c + 81z, 44b + 41y)
HCD spectrum of the intact
isotopic patterns of b
115
(light
esolution setting of 480,000
240,000, these two overlapping
resolved making still possible the
40
50
60
elative Abundance
1487.35107
R=115000
z=8
734.35059
R 170001
ering the resolution further, to
sly assign these two different
ody in denatured form
10
20
30
R
=
z=1
2703.31738
R=81100
z=4
3615.62646
R=81504
z=?
558.15320
R=194700
patterns of b
115
(light chain)
n setting of 480,000.
8+
FIGURE 9. ProSightPC software results of the EThcD analysis of the intact
f
500
1000
1500
2000
2500
3000
3500
4000
m/z
0
58.0
1558.5
1559.0
z=8
1558.52917
R=187104
z=8
1558.90479
R=177204
z=8
* * * * * * * *
*
*
* * * * * * *
Herceptin antibody in denatured orm.
01.61914
=140400
z=9
2963.13013
R=170604
z=?
3320.76929
R=172004
z=?
3646.76025
R=155701
z=1
(light chain)
(heavy chain)
3000
3500
4000
12450.14772
12448.96697
*
*
-2.9ppm
-2 3ppm
12445
12450
12455
.
(light chain) and b (heavy
+
8+
5
113
te mass spectrometer at three
Conclusion
Orbitrap top-down analysis can be used as a powerful tool for the
characterization of therapeutic mAbs in both native and denatured forms.
Ultrahigh resolving power achieved for 3 second detection time on a compact
3320
04
1558.77185
R=44904
1559.02856
R=59404
-
,
high-field Orbitrap instrument, allowed to baseline isotopically resolve an 148
kDa intact antibody.
Ultrahigh resolving power was used to resolve and unambiguously assign
overlapping isotopic clusters from top-down analysis of an intact antibody.
137
04
1558.78174
R=110904
1559.03113
R=114804
*
* * * *
* * *
8
References
1. Denisov
et al., Int. J. Mass Spectrom.,
2012, 325–327.
2 Lange
et al
Presented at the 59
th
ASMS Conference on Mass Spectrometry and
1559.0
917
04
1558.77966
R=189104
1559.03052
R=177804 1559.27942
R=99604
*
* * * *
* * *
b
115
(light chain)
*
b
113
(heavy chain)
*
+
8+
.
.,
Allied Topics, Denver, CO, June 5–9, 2011; Poster M093.
3. Shaw JB and Brodbelt JS,
Anal. Chem.,
submitted.
4. Horn
et al.,
Presented at the 60
th
ASMS Conference on Mass Spectrometry and
Allied Topics, Vancouver, BC, Canada, May 20–24, 2012; Poster W180.
ation of ETD and HCD (EThcD)
natured conditions (Figure 8 and
(1350 uscans) in a direct infusion
Acknowledgements
We would like to thank Jared Shaw from the University of Texas for fruitful discussions.
Herceptin is a trademark of Genentech Inc. Micro Bio-Spin is a trademark of Bio-Rad Laboratories, Inc. ReSpect is
a trademark of Positive Probability Ltd. 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 For research use only not for use in diagnostic procedures
ions (62c + 29z, 25b + 9y) from
+ 32y) from the heavy chain.
or the light chain and 23.7% for
%.
.
,
.
