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©2013 Thermo Fisher Scientific Inc. All rights reserved. HUMIRA is a trademark of AbbVie. ISO is a trademark of the International Standards
Organization. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Specifications, terms and pricing are
subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
Thermo Fisher Scientific,
San Jose, CA USA is
ISO 9001:2008 Certified.
BioPharma 2013_PN63712_E_11/13S
2000
2500
3000
3500
0
1705.5
1706.0
1706.5
1707.0
R=111081
R=105350
R=109093
R=104772
108541
R=110104
2
R=108646
R=109703
R=105375
R=99113
R=86808
Conclusion
•
The analysis of intact and reduced antibodies on the Orbitrap Elite
mass spectrometer provides the accurate molecular weight, as well as
valuable information about the presence and abundance of glycoforms.
•
Analysis of the reduced antibody provides isotopically resolved mass
spectra for both light and heavy chain.
•
The combination of multiple fragmentation techniques in top-down
analysis (SID, CID, HCD and ETD) generates comprehensive
sequence coverage and enables fast localization of modifications with
minimum sample preparation.
•
For measurements of intact light and heavy chain as well as for the
detection of fragment ion spectra from top-down experiments ultra-
high resolution as provided by the Orbitrap Elite mass spectrometer is
essential.
Abbreviations
ACN, acetonitrile; CID, collision-induced dissociation; C-trap, curved
linear trap; DTT, dithiothreitol; ETD, electron transfer dissociation; FA,
formic acid; HCD, higher energy collision-induced dissociation; mAb,
monoclonal antibody; µS, micro-scan; SID,
in-source
decay; SIM, single
ion monitoring.
References
1.
Bondarenko, P.V., Second, T.P., Zabrouskov, V., Makarov, A. &
Zhang, Z. Mass measurement and top-down HPLC/MS analysis of
intact monoclonal antibodies on a hybrid linear quadrupole ion trap-
Orbitrap mass spectrometer.
Journal of the American Society for
Mass Spectrometry
20
, 1415-24 (2009).
2.
Michalski, A.
et al.
Ultra high resolution linear ion trap Orbitrap mass
spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and
versatile peptide fragmentation modes.
Molecular & cellular
proteomics: MCP
(2011).doi:10.1074/mcp.O111.013698
Acknowledgements
We would like to thank Paul Thomas from Northwestern University (USA)
for processing the HUMIRA ETD data.
HUMIRA is a trademark of AbbVie. 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.
ge of the HUMIRA heavy chain
D, HCD, and ETD. Optimized
ssure settings. N: Putative
TD spectrum of
intact
HUMIRA
agment ion spectrum of
intact
olution possible.
200
400
1600
1800
2000
1395.24207
z=18
1507.50037
z=8
1321.86304
z=?
1594.27380
z=8
17.55786
z=?
1629.42053
z=8
1756.70886
z=7
IM scan mode (z=43). 60 µscans were
317 Da. The inserts on the right
tate detected at m/z 1185 and masses
1186
0
0
1185.4508
R=122004
1185.8925
R=109804
1186.2635
R=127904
Isotope pattern and average
mass of the
intact heavy chain
after deconvolution with Xtract
Monoisotopic mass (M) of the
intact heavy chain
after
deconvolution with Xtract
Deconvolution
50890
50900
50910
50920
50930
50940
m/z
0
20
40
60
80
100
0
20
40
60
80
100
RelativeAbundance
50891.04317
50922.07492
Humira_heavy_av_SIM
#
1
RT:
1556.88
AV:
1
NL:
3.30E3
T:
FTMS+ pESISIMms [1180.00-1190.00]
1185.2
1185.4
m/z
0
20
40
60
80
100
RelativeAbundance
1185.2190
R=117700 1185.2921
R=117704
1185.1287
R=120804
1185.3820
R=118304
1185.4961
R=120004
z=43
zoom
