Analytical Chemistry Chemical Cytometry Quantitates Superoxide

(pH 8.0) with cysteine and cystamine at concentrations of 6 and
1 mM, respectively. Isoform-A was enriched by incubating the
IgG2 under the same conditions but with the addition of 1.0 M
guanidinium chloride (GuHCl) to the buffer. The samples were
protected from light at 2-8 °C for approximately 48 h.
The IgG2 mAb#3 was subjected to Cysf Ser mutagenesis at
position 232 as described by Allen et al. 10 Site-directed mutagenesis
was performed using the QuickChange XL site-directed mutagenesis
kit (Stratagene, La Jolla, CA). The mutant was stably transfected into
a serum-free suspension-adapted CHO cell line. 31 Following production,
the mAb was purified by protein A affinity chromatography.
Incorporation of the expected mutation was verified on the purified
molecule by size exclusion chromatography (SEC) MS. 12
For IMMS analysis, IgG samples were buffer exchanged and
concentrated using Vivaspin 30 kDa molecular weight cutoff filters
(GE Healthcare, Buckinghamshire, UK). For experiments performed
under native conditions, the IgGs were diluted using 160 mM
ammonium acetate (pH not adjusted) to a final working concentration
of ∼3 µM.
Mass spectrometry experiments were performed using a
hybrid ion mobility quadrupole time-of-flight MS (Synapt, Waters
Inc., Milford, MA) equipped with a nanoelectrospray ionization
(ESI) source using metal coated borosilicate glass capillaries
(nanoflow probe tips, long thin walled, Waters Corporation).
Solution flow rates of ∼75 nL/min and an ESI capillary voltage of
∼1.3 kV were used for all experiments. The source temperature
was 50 °C, and the pressure of the vacuum/backing region was
3.5 mbar. Each ion mobility mass spectrum was acquired from
m/z 4000-8000 every 2 s; approximately 12 counts per scan were
observed. The signal was typically averaged for approximately 10
min. Gentle source conditions were used to minimize gas phase
unfolding of the protein (sample cone: 40 V, trap voltage: 10 V,
transfer lens: 12 V, bias: 25; cone gas: 30 L/Hr; P TRAP(Ar): 0.0175
mbar). Nitrogen was used as the mobility carrier gas, and the
following parameters were found to give optimal ion mobility
separation (PIMS: 0.5 mbar, wave velocity: 300 m/s, wave height:
9.8 V). The instrument was mass calibrated using a 50 µg/µL
CsI solution. Waters’ raw data files were translated to Matlab
binary files using software developed in-house. Data processing
and plotting were performed in Matlab (The MathWorks Inc.
Natick, MA) and Igor Pro (WaveMetrics Inc., Lake Oswego, OR).
RESULTS AND DISCUSSION
Antibodies belonging to the IgG2 subclass exist as a group of
distinct isoforms with different disulfide connectivities between
the Fab domain and the hinge region of the molecule. The goal
of this study was to investigate the ability of IMMS to successfully
separate these discrete IgG2 isoforms.
A nano-ESI ion mobility mass spectrum for a solution of 3 µM
mAb#1 (theoretical MW for the most abundant glycoform (G1F/
G1F): 149821.50 Da) in 160 mM ammonium acetate is shown
Figure 2a. A narrow charge state distribution centered on the 24+
ion is observed. Ion mobility spectra for all MAbs analyzed were
acquired with minimal acceleration voltages (sample cone: 40 V,
trap: 10 V, transfer: 12 V). These gentle tuning conditions were
found to provide optimal resolution in the ion mobility dimension
(31) Rasmussen, B.; Davis, R.; Thomas, J.; Reddy, P. Cytotechnology 1998, 28,
31–42.
Figure 2. Ion mobility TOF mass spectra of (a) mAb#1 and (c) mAb#2.
The normalized ion mobility intensities are graphed as contour plots with
9 levels from 4% to 100% intensity. Extracted arrival time distributions
for the 26+ charge states of (b) mAb#1 and (d) mAb#2.
(vide infra). However, with these conditions, the mass spectral
peaks are relatively broad making it impossible to observe the
individual glycoforms of the IgG. The glycoforms for these IgG
molecules can, however, be readily resolved using higher source
voltages (data not shown). Robinson and co-workers have previously
observed that optimal conditions for the mass and mobility
dimensions are often not compatible on the Synapt instrument
when analyzing large protein assemblies. 32
Ion mobility separation of mAb#1 (Figure 2a) reveals two distinct
conformer populations. For example, the mobility spectrum for the
26+ charge state (Figure 2b) shows two distinct peaks with a 1.1
ms difference in drift times (9.3 and 10.4 ms, respectively). Similar
distributions are observed for each charge state. However, with the
tuning conditions employed here, the best mobility resolution was
achieved for the 27+, 26+, and 25+ charge states. To elucidate if
the heterogeneity of the sugar chains on the antibody influences the
observed gas-phase conformers, deglycosylated mAb#1 was analyzed.
The same distribution of gas-phase conformers is revealed in the
ion mobility mass spectrum of mAb#1 with the sugars released
(Figure S1a, Supporting Information). This clearly demonstrates that
these multiple conformers are not caused by sugar heterogeneity
but instead are related to conformational differences in the protein
chains. The arrival time distributions for each charge state virtually
overlaps with that of the glycosylated protein. For example, the major
peaks in the arrival time distribution plot of the 26+ charge state of
deglycosylated mAb#1 are only shifted by -0.13 ms compared to
(32) Ruotolo, B. T.; Benesch, J. L. P.; Sandercock, A. M.; Hyung, S.-J.; Robinson,
C. V. Nat. Protoc. 2008, 3, 1139–1152.
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