Analytical Chemistry Chemical Cytometry Quantitates Superoxide

that of the glycosylated IgG (Figure S1b, Supporting Information).
This indicates that the glycosylated and deglycosylated antibodies
have nearly identical gas-phase collision cross sections.
The relative abundance of each gas-phase conformer can be
roughly estimated by fitting the arrival time distribution to a sum
of log-normal functions and integrating the relative area of each
peak. The log-normal function is an asymmetric Gaussian function
whose logarithm is normally distributed. 33 The log-normal function
was used because it was empirically found to best fit the
experimental peak shape of this ion mobility data. For the 26+
charge state, the normalized area of peaks 1 and 2 from the best
fits are 42% and 58%, respectively. Similar areas are observed for
other charge states. These abundances are similar to the relative
peak areas observed in electropherograms of IgG2s separated with
capillary electrophoresis. We and others have shown CE-SDS to
be a resolving technique for the separation of IgG2 structural
isoforms. 3,5,35 However, this correlation alone does not signify that
the resolved gas-phase conformers are definitively due to disulfide
variants.
To elucidate whether the gas-phase conformers observed for
IgG2 molecules are related to their disulfide connectivity, we
analyzed an IgG1 antibody, mAb#2 (theoretical MW for the most
abundant glycoform (G1F/G0F):148408.0 Da), as a control (Figure
2c). The most significant difference between human IgG1 and
IgG2 subclasses is the primary structure of the hinge region,
resulting in the absence of disulfide related isoforms in the IgG1. 3,4
In contrast to the IgG2 mobility data, for each charge state of
mAb#2, the arrival time profile is relatively narrow and consists
of a single uniform distribution (Figure 2c). For example, the
arrival time profile for the 26+ charge state of mAb#2 (Figure
2d) shows a single peak at 8.7 ms. This arrival time profile is
representative of the distribution observed for each of the charge
states. This suggests that the multiple conformers observed of
mAb#1 are due to disulfide variants in the antibody.
To further demonstrate that the observed gas-phase conformers
are indeed IgG2 disulfide variants, individual disulfide isoforms
were selectively enriched in a refolding experiment using redox
chemistry employing cysteine/cystamine. Dillon et al. previously
demonstrated that isoform IgG2-A and IgG2-B can be redoxenriched
by refolding with and without 1 M GuHCl, respectively. 4
Under redox conditions in buffer alone, IgG2-A is refolded to IgG2-
B. Liu et al. demonstrated that a slow conversion of IgG2-A to
IgG2-B also occurs in vivo. 34 Isoform conversion toward IgG2-A
requires in vitro refolding in presence of low levels of chaotropic
reagents. 4 Figure 3a,b shows the arrival time distributions for the
+26 charge state for redox enriched mAb#1 in the presence of
guanidine (isoform A) and redox enriched mAb#1 in the absence
of guanidine (isoform B), respectively. In contrast to IMMS for
the untreated Mab#1 antibody (Figure 3, dashed line), a single
abundant conformer is observed for each of these enriched
isoforms. As a control, the IgG1 antibody, mAb#2, was also
subjected to the same redox refolding protocol with or without
guanidine. All treated IgG1 samples have identical arrival time
(33) Brown, R. Personal Eng. Instrum. News 1991, 8, 51–54.
(34) Liu, Y. D.; Chen, X.; Enk, J. Z.; Plant, M.; Dillon, T. M.; Flynn, G. C. J. Biol.
Chem. 2008, 283, 29266–29272.
(35) Lacher, N. A.; Wang, Q.; Roberts, R. K.; Holovics, H. J.; Aykent, S.; Schlittler,
M. R.; Thompson, M. R.; Demarest, C. W. Electrophoresis 2010, 31, 448–
458.
6754 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
Figure 3. Arrival time distributions for the 26+ charge states of IgGs
treated with redox reagents (cystamine, cysteine). (a,b) mAb#1 and
(c,d) mAb#2 (control). Samples represented in (a) and (c) had 1 M
GuHCl added to the buffer. The dashed line shows the arrival time
distribution for the 26 + charge state of untreated mAb#1.
distribution profiles as the untreated IgG1 molecule (Figure 3c,d),
demonstrating that this refolding protocol does not affect the
overall tertiary structure of the antibody. Comparing the untreated
IgG2 IMMS trace with the enriched isoform distributions (Figure
3a,b) identifies peaks 1 (9.3 ms) and 2 (10.4 ms) in the mobility
spectra as isoform A and isoform B, respectively. The relatively
late arrival time of isoform B indicates that this form of the
antibody has a larger gas-phase collision cross section compared
to isoform A. The IMMS resolution of the isoforms correlates with
the previously observed capillary electrophoresis separation. 5,8,35
With CE, IgG2 isoforms were resolved into two peaks, with IgG2-A
migrating more rapidly than the IgG2-B isoform. The intermediate
IgG2-A/B forms were split between the two peaks, IgG2-A/B1
migrating with -A and IgG2-A/B2 with -B. 8
While the disulfide connectivities of IgG2 isoforms have been
well characterized, 3,4,8,9 only limited information is available
describing the overall tertiary structure of human IgGs. To
investigate if the relative ordering of gas-phase collision cross
sections (IgG1 ≈ IgG2-A < IgG2-B) correlates with IgG solution
structures, collision cross sections were calculated for two IgG
structures using Waters’ CCS software. 36 The calculated cross
section of an IgG antibody, with a disulfide bonding pattern
consistent with the B isoform, is 8385 Å 2 (unpublished results).
This value is 3% smaller than the calculated cross section of
an IgG1 antibody (protein data bank code 1HZH, 8653 Å 2 ). 37
(36) Williams, J. P.; Lough, J. A.; Campuzano, I.; Richardson, K.; Sadle, P. J.
Rapid Commun. Mass Spectrom. 2009, 23, 3563.
(37) Saphire, E. O.; Parren, P. W.; Pantophlet, R.; Zwick, M. B.; Morris, G. M.;
Rudd, P. M.; Dwek, R. A.; Stanfield, R. L.; Burton, D. R.; Wilson, I. A. Science
2001, 293, 1155–1159.