5
Figure 4. Deconvoluted mass spectrum of rituximab with annotated glycoforms (top) and
comparison of theoretical and measured masses for the five most abundant glycoforms (table)
Figure 5. Full MS spectra acquired from 1 ng intact rituximab, applying increasing settings of source CID (SID)
For the acquisition of the full MS spectrum of the intact
antibody, the optimum setting of the source CID was
evaluated (Figure 5). This setting was found to be crucial
in obtaining a high quality spectrum. The application of
25–90% source CID is beneficial for most proteins. For
this sample the optimum setting was 80% SID.
146925.606
146619.406
148010.563
162 Da
162 Da
162 Da
162 Da
162 Da
162 Da
146619.406
Man5/Man5
(G1F/G2F)SA1
(G1F/G2F)SA2
G1F/G2FSA2
G1F/G2FSA1
146541.406
146925.606
Man5/Man5
(G2F/G2F)SA2
+2740.35
+2673.54
+2513.07
(G2F/G2F)SA1
G0F/G0F
G0F/G1F G0F/G2F or
(G1F)
2
G1F/G2F
G2F/G2F
Measured
147,075.969
147,237.734 147,399.516 147,561.766 147,721.813
Theoretical
147,074.985
147,237.126 147,399.267 147,561.408 147,723.549
Δ ppm
-6.7
-4.1
-1.7
-2.0
11.8
0
5
10
15
20
Time (min)
0
20
40
60
80
100
Relative Abundance
12.24
12.55
4.78
5.49
14.06
11.89
16.56
21.80
6.82
0.60
2000
2500
3000
3500
4000
4500
5000
m/z
0
20
40
60
80
100
Relative Abundance
3190.65
3309.78
2979.82
3567.96
3656.31
2714.43
1940.42
BPC
SID 40%
SID 50%
SID 60%
SID 70%
SID 80%
3841.98
2304.46
3968.41
4224.07
4492.29
2000
2500
3000
3500
4000
4500
5000
m/z
0
20
40
60
80
100
Relative Abundance
2949.09
3145.74
2784.44
3285.71
2304.07
3526.92
4055.44
4543.99
2000
2500
3000
3500
4000
4500
5000
m/z
0
20
40
60
80
100
Relative Abundance
3009.29
2888.03
2779.08
3347.44
2678.07
3506.87
2457.72
3875.57
4497.14
2000
2500
3000
3500
4000
4500
5000
m/z
0
20
40
60
80
100
Relative Abundance
3133.74
2945.78
3347.37
2888.02
3506.68
3682.01
2730.67
3980.61
2496.66
4463.02
2000
2500
3000
3500
4000
4500
5000
m/z
0
20
40
60
80
100
Relative Abundance
3272.96
3068.44
3506.69
3005.83
3681.99
2832.48
3875.75
2587.05
4207.88
