Journal of the American Chemical Society
Article
membrane, suggesting it retained antibody-recognition activity,
whereas no accumulation of labeled trastuzumab was observed
in HER2-negative cells (Figure S36). The sites oxidized under
this trastuzumab labeling condition were detected by MS in
the same manner as in Figure S20, and histidine, tryptophan,
and tyrosine residues were detected as oxidation sites. There
was good agreement between the oxidized sites and labeled
Subsequently, we identified which amino acid residues were
labeled in the Fc region by mass spectrometry. The SDS-PAGE
band corresponding to the heavy chain of Cy3-labeled
trastuzumab was digested in a gel with trypsin, and the
resulting peptide fragments were analyzed by LC-MS. Three
labeling sites were identified by the detection of Cy3-labeled
The protein A mimic was reported to bind not only to
human IgG but also to rabbit IgG.39 Rabbit polyclonal anti-V5
epitope was successfully labeled using the beads (Figure S44).
Finally, we performed selective labeling of the Fc region while
purifying the antibody from rabbit serum. Rabbit serum was
mixed with beads to purify the antibody, followed by a labeling
reaction. The antibody Fc region was selectively modified
while enriching the antibody from the serum protein mixture
CONCLUSIONS
■
We found that MAUra efficiently labeled histidine residues
under 1O2 generation conditions. To our knowledge, this is the
first report of a histidine-selective nucleophilic protein labeling
1
reagent targeting oxidized histidine by O2. Utilizing the short
diffusion distance of 1O2, we demonstrated antibody Fc-
selective labeling on magnetic beads functionalized with a
ruthenium photocatalyst and Fc ligand, ApA. This method has
the following advantages: (1) protein functionalization with a
short reaction time of 5 min, (2) one-pot operation of both
antibody enrichment and labeling, (3) application to all types
of IgG (human and various animal IgGs), and (4) reusability
of the beads. Three histidine residues located around the ApA
binding site were identified by LC-MS analysis, suggesting that
1O2-mediated histidine labeling can be applied to a proximity
labeling reaction on the nanometer scale. Although the
distance between the catalyst and the ligand was not precisely
controlled on the bead surface in this study, future improve-
ments in site selectivity and efficiency can be expected by
designing reaction fields with homogeneous ligand−catalyst
distances. Research into further applications, such as intra-
cellular photocatalyst-proximity labeling, is currently in
progress.
Figure 4. Identified labeling sites. (A) LC analysis of labeled peptide
fragments. (B) 3D structure of trastuzumab Fc region, labeled
histidine residues (red), and ApA binding site (green) (PDB: 3D6G).
The surface is displayed for one chain. (C) Table of distance from the
ligand-binding site and side chain solvent accessibility of histidine
residues in the Fc region of trastuzumab. See Figure S42 for distance
calculations. Side chain solvent accessibilities (Å2) were estimated by
Discovery Studio 4.1 and in X-ray crystal structure of the trastuzumab
Fc region (PDB: 3D6G).
EXPERIMENTAL SECTION
■
Peptide Labeling Using a Photocatalyst. Labeling reagent
(from a 100 mM stock solution in DMSO, final concentration 1 mM)
and photocatalyst (from a 100 mM stock solution, final concentration
1 mM) were added to a solution of peptide (final concentration 100
μM or 1 mM) in 50% CH3CN solution in 10 mM MES buffer (pH
7.4) in a 1.5 mL tube. The solution was distributed and irradiated
with white light (white LED, ISL-150 × 150 H3WH4R, 8.0 0.5
mW/cm2, CCS Inc.) for 10 min on ice. The resulting solution was
desalted using C18 pipet tips (Nikkyo Technos Co., Ltd.). The
reaction mixture was diluted 10 times with 0.1% trifluoroacetic acid
(TFA) and mixed with α-cyano-4-hydroxycinnamic solution (5.0 mg/
mL solution in acetonitrile/0.1% aq TFA = 0.5 μL/0.5 μL), and the
mixture was placed on a MALDI-TOF plate and dried at room
temperature. The labeled peptides were detected by MALDI-TOF
MS analysis (Bruker, UltrafleXtreme, or ABSCIEX TOF/TOFTM
5800).
MS analysis after digestion of 2-labeled trastuzumab with
trypsin and Glu-C revealed three labeling sites: H288, H313,
and H436 (Figure 4B and Figure S41). Based on the positional
relationship between the labeled sites and reaction field on the
beads, labeling efficiencies were presumed to depend on both
the distance from the ApA-binding site and the surface
exposure level (Figure 4C). The labeling of Tyr439, the
tyrosine residue closest to the ApA binding site, was not
detected. It is probable that Tyr439 is buried beneath His436,
and the ruthenium photocatalyst is in an environment where
His436 can be preferentially approached. Other tyrosine
residues located far from the ApA binding site (>21 Å) with
low solvent accessibility were also not labeled (Figure S43).
This technique should be applicable to various IgG
antibodies, as the Fc region has a conserved sequence present
in all types of IgG. Cetuximab and rituximab, humanized
antiepidermal growth factor receptor (EGFR), and anti-CD20
antibodies were also labeled using the same method. As
expected, the beads selectively labeled the Fc regions of the
antibodies (Figure S44). Using rituximab, the binding sites
were identified by nanoLC-MS/MS in the same manner as in
the experiment with trastuzumab. Two histidine residues
(H289 and H437 in rituximab) corresponding to trastuzumab
Bead Preparation. Dynabeads (Dynabeads MyOne carboxylic
acid, 400−800 nmol/mg carboxylate, Invitrogen, 4.0 mg) were
washed with dimethylformamide (DMF) and dissolved in 800 μL of
200 mM N-hydroxysuccinimide (NHS)/DMF solution. EDCI·HCl
(final concentration 200 mM) was added to the mixture, which was
stirred for 2 h at room temperature, and the beads were washed with
DMF to give NHS ester-functionalized Dynabeads (stored in
isopropyl alcohol at −20 °C). The NHS ester-functionalized
Dynabeads (1.0 mg) were washed three times with 200 μL of
DMF. Amine-conjugated Ru/dcpby (from 10 mM solution in H2O,
final concentration 100 μM), amine-conjugated Fc ligand (from 10
mM solution in DMF, final concentration 100 μM), and NEt3 (from
100 mM stock solution in DMF, final concentration 1 mM) (final
concentration of beads: 5.0 mg/mL) were added and stirred at room
temperature for 1 h, and the beads were washed three times with
D
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX