10
In an attempt to further confirm the sequences of the light
and heavy chains, two types of top-down experiments
were performed: all-ion fragmentation (AIF) with
fragmentation upon collision in the HCD cell and a
multiplexed (5-plex), targeted MS
2
experiment on five
selected charge states each of the light and heavy chains.
All spectra were acquired at 70,000 resolution. For the
targeted MS spectrum, a retention-time-dependent mass
list was used, targeting first the earlier eluting light chain
(RT 13.16 min:
m/z
1536.96, 1646.6, 1773.3, 1920.7,
2095.4) and later the heavy chain (RT 16–20 min:
m/z
1584.6, 1635.7, 1684.7, 1748.5, 1810.9). In this type
of experiment, the first charge state listed on the inclusion
list is selected and sent to the HCD cell for fragmentation.
The product ions are stored in the HCD cell while the
second charge state is isolated, sent to the HCD cell,
fragmented, and stored in the cell until the fifth charge
state has also been fragmented. All ions from the five
individual isolation and fragmentation steps are sent
together to the Orbitrap analyzer, resulting in one single
fragment ion spectrum.
The fragment ion assignment for the light chain is
displayed in Figure 10. There is good coverage of both the
N- and C-terminal ends as well as some fragments in the
center of the sequence, resulting in 28% coverage,
respectively 15% of the theoretical fragments. For the
heavy chain, fragmentation was less efficient with both
methods and resulted in about 20 fragments, most of
which represent the sequence termini.
To further confirm the sequences, a bottom-up approach
was performed using a digest with trypsin following
reduction and alkylation of the antibody. The chrom-
atogram obtained from the digest is displayed in
Figure 11. A database search against a four-entry database
containing the light chain, both variants of the heavy
chain, and trypsin revealed a sequence coverage of the
light chain of 96% and for the heavy chain of 78.8%
(Figure 12). The two short missing peptides from the light
chain (LEIK and EAK) could be detected as intact masses
only in the full MS spectra, whereas the peptide EAK was
identified based on the accurate mass corresponding to the
peptide containing a missed cleavage
EAK
VQWK. Taking
into account the peptides identified based on MS/MS
spectra and based on accurate masses of the small intact
peptides, sequence coverage for the light chain is 100%.
Figure 10. Matched sequence coverage of the rituximab light chain based on fragment ions obtained from AIF experiments. Seventeen b- and 50 y-ions
were assigned, corresponding to 15.7% of the theoretical number of fragments (67 of 426).
