Supporting Information:
Native MS and ECD Characterization of a Fab-Antigen
Complex May Facilitate Crystallization for X-ray
Diffraction
Ying Zhang, Weidong Cui, Aaron T. Wecksler, Hao Zhang, Patricia Molina, Galahad Deperalta, and
Michael L. Gross*
Detailed Methods and instrumentation
Chemicals
Trifluoroacetic acid (TFA), formic acid (FA), urea, dithiothreitol (DTT), and all HPLC-grade solvents (H2O and acetonitrile,
ACN) were obtained from Sigma-Aldrich (St. Louis, MO) at the highest purity available unless stated otherwise. Protein samples,
including the free VEGF, Fab-1, and the complex, were provided by Protein Analytical Chemistry, Genentech Inc.
Protein and Protein Complex Separation
The Fab-1-VEGF complex was prepared with a 2:1 molar ratio of Fab-1:VEGF and incubated at 37 °C for 15 minutes. The Fab1:VEGF complex and remaining unbound Fab-1 were purified by size exclusion chromatography (SEC), performed on an
Agilent 1100 HPLC system using a Tosoh-Bioscience SEC TSKgel G2000SWXL (7.8 x 300 mm, 5 μm) column. Isocratic
runtime was 30 min at 0.5 mL/min using the mobile phase buffer (0.2 M K2HPO4, 0.25 M KCl, pH 6.2) at ambient temperature.
Samples of 500 μg Fab-1:VEGF mixtures were fractionated and collected by monitoring at 280 nm, and the purity of the collected
complex and Fab-1 were re-analyzed with 20 μg injections of purified substance. Data were analyzed using Chromeleon Software
package (Thermo Scientific, CA).
Protein Buffer Exchange
VEGF, Fab-1 and the complex samples were buffer-exchanged into 200 mM ammonium acetate (pH 6.8) by using a molecularweight cutoff filter (GE Healthcare Bio-Sciences, PA) according to their sizes. For one cycle, each sample was centrifuged at
10,000 rpm for 5 min. Each sample was washed for at least 10 cycles for better desalting prior to direct injection into MS.
Mass Spectrometry
Both native ESI and top-down experiments were performed on a Bruker SolariX 12 T FT-ICR mass spectrometer (Bruker,
Bremen, Germany), as described previously [1]. In summary, samples for native ESI (5-10 uL) were loaded into custom-made,
off-line nano electrospray tips. The instrument was setup for direct infusion with the Bruker nano electrospray source, and
parameters were optimized by using model protein complexes ranging from ~60 to 150 kDa. All peaks observed in native ESI
were selected for fragmentation to optimize product spectrum.
The denaturing ESI was done by using a reversed-phase LC coupled with mass spectrometry, as described in a previous
publication[2]. In detail, the protein samples (both VEGF and Fab-1 separately) were denatured by 8 M urea and reduced by 10
mM DTT, and incubated at 37 °C for 30 min. An aliquot (10 uL) of the reduced protein samples were then mixed with 40 uL
H2O containing 0.1% FA, and were injected into a 50 uL loop and eluted onto a C4 analytical column (2.1 mm x 15 cm, BEH300,
Waters, MA) by an Agilent HPLC 1100 (Agilent, CA). Proteins were then separated with a linear gradient of 4% - 40% ACN,
0.1% FA, over 5 min by an Agilent HPLC 1200 (Agilent, CA). The ESI mass spectra were acquired in the positive-ion mode of
a MaXis 4G quadrupole time-of-flight (Q-Tof) mass spectrometer (Bruker, Bremen, Germany).
Data Analysis
To identify the fragment ions (MW range 200 to 1500), we utilized Biotools software (v3.2, Bruker, Bremen, Germany) where
the experimental masses measured at a resolving power of 130,000 (m/z 400) were submitted. In studies of this nature, the
primary structure was known, allowing the algorithm to calculate the accurate masses of all possible fragment ions. The
calculated masses of all b, y, c, and z ions were compared with the experimental masses, and assignments were accepted if the
experimental mass was within 5 ppm of theory. All peaks had signal-to-noise ratio greater than 5:1 for the assignment. No
modifications were considered because there were no glycosylations in this Fab, and post-translational modifications (e.g.,
oxidation and deamidation) were of low probability. It was not necessary to consider modifications typically introduced in
bottom-up experiments (e.g., Cys alkylation), an advantage of top-down. All assignments were manually validated.
Figure S1. Native ESI spectrum of free VEGF. Note that the native state of VEGF shows as mass of a dimer. Charge state is shown for the
most abundant peak.
Figure S2. Native ESI spectrum of free Fab. Charge state is shown for the most abundant peak.
Figure S3. Denaturing ESI mass spectrum of HC of the free Fab. Note the small peaks at m/z 700-1500 that originate from the LC, which is
not separated completely from the HC by reversed-phase HPLC. Charge state is shown for the most abundant species.
Figure S4. Denaturing ESI spectrum of LC of the free Fab. Charge state is shown for the most abundant species.
Figure S5. Denaturing ESI spectrum of the free VEGF. Charge state is shown for the most abundant species.
y4
b4
b5
b6
b7
Fab
Int.
b4
HC
LC
200
300
400
500
600
700
800
m/z
900
1000
1100
1200
Figure S6. Zoom-in region of top down mass spectrum using ECD, in concert with CAD and IRMPD (Figure 1B, native ESI of the complex)
of m/z 200-1250. The assigned fragments (b and y ions) are highlighted in different colors according to their source.
References
1.
2.
Zhang, H., Cui, W., Wen, J., Blankenship, R.E., Gross, M.L.: Native electrospray and electron-capture dissociation
fticr mass spectrometry for top-down studies of protein assemblies. Anal Chem 83, 5598-5606 (2011)
Zhang, Y., Rempel, D.L., Zhang, H., Gross, M.L.: An improved fast photochemical oxidation of proteins (fpop)
platform for protein therapeutics. J Am Soc Mass Spectrom 26, 526-529 (2015)