5
Thermo Scientific Poster Note
•
PN-64092-ASMS-EN-0614S
Conclusion
The monoclonal antibody c
and Orbitrap Fusion mass s
With optimized experiment
of the example monoclonal
mass error using Orbitrap F
67% fragment coverage wa
Elite with LC-MS analysis.
22% fragment coverage wa
Orbitrap Fusion MS with inf
Acknowledgeme
We would like to thank NIST for p
All trademarks are the property of Thermo Fi
This information is not intended to encourag
intellectual property rights of others.
FIGURE 6. Example mAb heavy
and HCD (red, 15%) using Orbit
f mAb triplicate runs were used
re 2 shows the results of this
similar between the three runs.
experiment condition. The
mass error for the analyzed
y identified. The results showed
ptimized experiment condition
Denatured_DTT_reduced_waters_5
11/27/2013 3:38:18 AM
RT:
0.00 - 10.00
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
4.36
4.38
4.44
4.47
3.61
4.50
4.58
4.65
4.75
4.88 5.50 5.49
3.83
5.76
0.16
6.04 6.32
0.34
1.08
7.69
0.86
7.02
1.22
7.59
1.56 1.75
7.76 7.95
9.99
2.03
8.22
9.81
8.75 9.20
2.12
3.37
2.97
2.61
NL:
1.34E9
TIC MS
Denatured_
DTT_reduc
ed_waters
_5
600
700
800
900
1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400
m/z
0
20
40
60
80
100
0
20
40
60
80
100
RelativeAbundance
897.1600
z=? 968.9456
z=?
835.4048
z=?
1100.8636
z=?
1274.5756
z=1
1345.3232
z=?
781.5663
z=2
2017.4332
z=?
1513.3986
z=?
734.2306
z=?
1862.4284
z=?
1614.2598
z=?
2200.7980
z=?
1729.4076
z=?
2276.3028
z=?
983.2181
z=?
1114.1092
z=?
908.7955
z=?
1249.2002
z=?
1319.2212
z=?
1432.1613
z=?
847.1902
z=?
1561.2941
z=? 1665.2634
z=?
796.0924
z=?
1789.7590
z=?
1937.6242
z=?
2134.7608
z=?
2315.2185
z=?
684.2031
z=?
NL:1.57E6
Denatured_DTT_reduced_wa
ters_5#1050-1185 RT:
3.57-3.83 AV:136T:FTMS
+pESIFullms
[400.00-4000.00]
NL:3.00E5
Denatured_DTT_reduced_wa
ters_5#1377-1708 RT:
4.25-4.89 AV:332T:FTMS
+pESIFullms
[400.00-4000.00]
Top-down ETD MS/MS
Top-down HCD MS/MS
Top-down ETD MS/MS
Top-down HCD MS/MS
Light chain
Heavy chain
Full MS of light chain
Full MS of heavy chain
AV:
73
NL:
8.39E6
3000
3200
3400
.5170
2968.7239
3029.3229
3092.4305
3158.1167
3226.7031
3298.4008
3451.8322
3533.8011
AV:
23
NL:
3.15E7
3000
3200
3400
3600
2
.5292
2968.6720
3029.2848
3092.3369
3158.0653
3226.7574
3294.8952
3451.9510
AV:
41
NL:
2.12E7
3000
3200
3400
3600
.5491
2968.7034
3029.2853
3092.3923
3158.1221
3226.8235
3298.4156
3451.7799
00
3200
3400
3600
3800
4000
795
29.2457
z=?
3092.3571
z=?
3158.1075
z=?
3226.8306
z=?
3373.3410
z=?
3537.9001
z=?
3808.9230
z=?
3941.2739
z=?
2770
2780
2790
2800
2810
2800.7177
z=?
2803.8044
z=?
2797.7166
z=?
2806.7508
z=?
2793.8027
z=?
2810.0054
z=?
82
2773.4383
z=?
2790.9592
z=?
ppm
2 ppm
Top-down LC-MS/MS of mAb light and heavy chain
To characterize the mAb sequence, top-down LC-MS/MS analysis was performed for
reduced mAb light and heavy chain with both ETD and HCD. Figure 4 a) is the
workflow diagram for the analysis. ETD and HCD MS/MS spectra were acquired in
two separate LC-MS runs. The molecular mass of both the light and heavy chains
were measured with low ppm mass accuracy (data not shown). ETD with a reaction
time of 15 ms generated the most fragmentation coverage with 52% of light chain
backbone sites fragmented. An HCD collision energy of 18% yielded the most
fragmentation with a fragmentation coverage of 39%. The combination of the ETD
and HCD data produced around 67% fragmentation coverage (Figure 5).
d experiment conditions, 100ng
F
F
2F
nvoluted spectrum of analyzed mAb
Figure 4 Top-down LC-MS/MS of mAb light and heavy chain
a) The online top-down MS/MS of NIST mAb light and heavy chain analysis with
both E D and HCD
FIGURE 5. Top-down LC-MS/MS of NIST mAb light chainusing Orbitrap Elite.
Sequence coverage from ETD (blue) and HCD (red) using Orbitrap Elite.
Deglycosylated and reduced mAb
ms ETD reaction time and 14% H
and HCD fragmentation spectra.
achieved combining both ETD an
