LETTER
Regioselective Labeling of Antibodies through
N-Terminal Transamination
Rebecca A. Scheck and Matthew B. Francis*
Department of Chemistry, University of California, Berkeley, California 94720-1460, and Materials Sciences Division,
Lawrence Berkeley National Labs, Berkeley, California 94720
B
ecause of their evolvable affinity for
both natural and synthetic epitopes,
monoclonal antibodies (mAbs) (1)
have attained a centrally important role as
imaging agents (2), targeting groups for
therapeutic delivery (2), components of
diagnostic arrays (3), and catalysts (4).
Many of these applications depend critically on the ability to add new function
through the covalent attachment of fluorophores, anticancer drugs, enzymes, and
nanoparticles (5–7). Typically, these groups
are attached to random locations on the antibody surface through the modification of
lysine, tyrosine, aspartate, and glutamate
residues. Although the function of the resulting conjugates may still be intact, the nonspecific nature of the modification leads to
product mixtures that may not be compatible with the intended application.
Antibodies consist of two identical light
chains and two identical heavy chains held
together by a series of disulfide bonds
(Figure 1, panel a) (8). The site-selective
modification of antibodies is particularly difficult to achieve due to the diverse and unpredictable set of amino acids in the hypervariable binding loops (Figure 1, panel b). As
one solution to this problem, numerous applications have targeted aldehydes that can
be introduced through the oxidation of carbohydrates on the Fc domain with NaIO4
(5, 9). Alternatively, the interchain disulfides
have been alkylated following reduction
(10, 11). Recombinant techniques can be
used to express the binding regions as a
www.acschemicalbiology.org
single polypeptide chain fused to a functional enzyme (12, 13).
As a simple alternative that can be used
to label antibodies from virtually any source,
we have applied a biomimetic transamination reaction that introduces ketone groups
on the N-termini (Figure 2, panel a) (14–19).
This reaction occurs upon exposure to
pyridoxal 5=-phosphate (PLP) at 37–50 °C
in buffered aqueous solution. The resulting pyruvamide derivatives can be elaborated easily through oxime formation with
functionalized alkoxyamines (20–23). A
key advantage of this strategy is its selectivity for the N-terminal amino group
(i.e., lysine residues do not participate)
(14), thus affording antibody conjugates
that are labeled in a limited number of
locations.
Our studies toward the modification of
these locations were conducted with monoclonal mouse anti-FLAG IgG (1) and the corresponding Fab fragments (2) obtained after proteolysis with papain. Samples were
incubated with 10 mM PLP in pH 6.5 phosphate buffer for 18–20 h to afford ketone
4. After removal of excess PLP via centrifugal ultrafiltration, the sample was labeled
with fluorescent alkoxyamine 6. Analysis by
SDS-PAGE revealed fluorescent bands only
in samples that had been treated with PLP
(Figure 2, panel b). Higher conversion was
observed when the protein was incubated
with PLP at 50 °C, leading to fluorescent labeling of both the heavy (50 kDa) and light
(25 kD) chains. The reaction was equiva-
A B S T R A C T A convenient new method is described for the introduction of ketone groups at
the N-termini of antibodies. The reaction occurs
in the presence of pyridoxal-5=-phosphate under
conditions mild enough to maintain antigen
binding function, as confirmed by enzyme-linked
immunosorbent assay. Further derivatization of
these functional sites was accomplished
through oxime formation, yielding well-defined
antibody conjugates for a wide range of applications. The ability of the modified antibodies to
bind their targets was confirmed via immunodot
blot analysis. The generality of this method has
been demonstrated on a number of monoclonal
and polyclonal antibodies, all with different
binding specificities.
*Corresponding author,
[email protected]
Received for review September 14,
2006 and accepted March 29, 2007.
Published online April 13, 2007
10.1021/cb6003959 CCC: $37.00
© 2007 American Chemical Society
VOL.2 NO.4 • ACS CHEMICAL BIOLOGY
247
a
b
Two identical
Fab fragments
(2)
Papain digest
(1) IgG
Gln1
+
One Fc fragment
(removed)
Asp1
that this site can be modified with smaller alkoxyamines (see Figure 2,
panel b). When the identical
reaction sequence was carried out on Fab
(2), a single new species was observed by
nonreducing SDS-PAGE (20% at 37 °C and
42% at 50 °C, Figure 2, panel c). Upon exposure to DTT, the PEG-modified light chain
could not be resolved from the heavy chain.
No polymer conjugation was seen for the
heavy chain.
Because the N-termini of IgG and Fab are
in proximity to their antigen-binding regions,
we sought to confirm that normal epitope
recognition was not disrupted. Two complementary immunoassays were used to do
this: a sandwich enzyme-linked immunosorbent assay (ELISA) and an immunodot blot.
For the ELISA, modified and unmodified
anti-FLAG antibody samples were first passively bound to a 96-well plate. Exposure of
these wells to bacterial alkaline phosphatase (BAP) displaying the FLAG peptide
was followed by incubation with anti-BAP
antibodies conjugated to horseradish peroxidase (HRP). Colorimetric substrate addition yielded the same signal for antibodies
activated with PLP as was obtained for their
unmodified counterparts (Figure 4, panel a).
We subsequently explored a dosedependent study of IgG samples that were
modified at 50 °C. If their antigen recognition capacity was diminished, a systematic
loss of signal would be expected as concentrations were lowered. However, antibodies
with or without exposure to PLP exhibited
identical binding over the full range of concentrations investigated (Figure 4, panel b).
We thus conclude that the PLP modification
reaction itself has minimal effects on antigen binding capabilities.
To explore whether subsequent oxime
formation perturbed antigen binding, a
sandwich ELISA was also performed for Fab
samples that were treated with commercially available biotin alkoxyamine 8 following reaction with PLP (Figure 4, panel c). As
Figure 1. Antibody structure. a) The structures of mouse IgG (1) and Fab (2) are shown with the light chains depicted in blue and the heavy chains in gray. The amino acid residues in the hypervariable regions are rendered
in yellow, and the N-termini are shown in red. Two identical Fabs are prepared via papain digest of IgG. b)
Close-up view of the antigen binding site, indicating the locations of the two N-termini.
lently successful for Fab, showing modification only after treatment with PLP.
The progress of the reaction was also
tracked by mass spectrometry (MS) analysis. Despite the small amount of inhomogeneity in the starting sample, the mass of Fab
(2) could be identified at 47,848 amu, in addition to a minor species at 47,976 amu
(Figure 3, panel a). After exposure to PLP,
two predominant new species were observed (Figure 3, panel b). The first, at
47,803 amu, corresponded to a transamination and concomitant decarboxylation of an
N-terminal aspartate, a residue that commonly occurs on the light chain of mouse
IgG. (For examples of mouse IgG antibodies
containing N-terminal aspartate residues on
the light chain, see PDB ID 1A3R, 1IGT,
1ACY, 1BQL, 1CKO, 1DBA, 1E4W, 1F11,
1F58, and 1FB1.) We have previously observed this behavior with peptide substrates
possessing this residue in this position
(14). Light chain specificity of this mass
loss was confirmed after reduction with
TCEP (Supplementary Figure 2). In addition
to this species, a small amount of a higher
molecular weight adduct could be detected
at 48,051 amu, corresponding to the addition of a single PLP molecule to the
N-terminal ketone (possibly through an aldol reaction). Upon addition of benzylalkoxyamine, both of these activated species were completely converted to the singly modified products at 47,908 and 48,156
amu, respectively (Figure 3, panel c). The
product resulting from oxime formation of
the transaminated heavy chain terminus
was observed at 47,954 amu, and the products resulting from the labeling of both
N-termini were observed at 48,014 and
47,264 amu. Analogous mass changes
could also be seen for the minor Fab species starting at 47,976 amu. Efforts are underway to confirm the identity of the
248
VOL.2 NO.4 • 247–251 • 2007
N-terminal PLP adduct and to suppress its
formation; nonetheless, this species appears to react with alkoxyamine reagents
and thus does not prevent labeling.
Although these spectra confirmed that a
single modification had occurred on each
chain, further evidence of the site specificity was obtained through proteolytic digest
analysis. After exposure of the samples to
trypsin, a single species was observed to
undergo the same pattern of decarboxylation followed by oxime formation (Figure 3,
panels d–f). The starting peak at 2544 m/z
was fragmented using tandem MS/MS to
yield a peptide sequence that matched
closely with other N-terminal sequences for
mouse immunoglobulin light chains [Examples of protein sequences of this type
can be found under NCBI accession numbers AAD34833 (identity 87%; positive
100%) AAB34860 (identity 87%; positive
100%), and PH1037 (identity 87%; positive
100%)] (Supplementary Figure 3). After
treatment with PLP, MS/MS analysis of the
modified peak (2499 m/z) indicated that
the loss of 45 m/z (Supplementary Figure 4)
had occurred from the N-terminal aspartate
residue, as seen in the b ion series (e.g., the
shift of the species at 328.23 m/z to 283.20
m/z). No peaks corresponding to PLP additions were identified by MALDI TOFMS.
We were able to quantify conversion to
the oxime product through the use of a previously reported poly(ethylene glycol) (PEG)
alkoxyamine (7) (24). Densitometry analysis
after SDS-PAGE and Coomassie staining indicated that for a transamination reaction
performed at 50 °C, 47% conversion was
achieved for the light chain of IgG (Figure 2,
panel c). As expected, lower conversions
were obtained at lower temperatures (25%
at 37 °C). Only a small amount of PEG attachment was observed for the IgG heavy
chain, although fluorescence data indicate
SCHECK AND FRANCIS
www.acschemicalbiology.org
LETTER
Light chain
Coomassie
Heavy chain
Coomassie
Fluorescence
before, no change in signal was
a
b
10−20% gel
1 or 2
observed. Biotinylated antibody
75 kD
R
2.5 mg/mL
H
samples were also used to asN Protein (16 µM for IgG)
50 kD
H2N
sess antigen-binding via an im37 kD
O
R = −CH2CO2H, −(CH2)2CO2H
munodot blot. SDS-PAGE and
37 or 50 °C, 18−20 h
25 kD
Western blot analysis confirmed
25 mM phosphate buffer
H
O
pH 6.5
75 kD
selective biotinylation of only
HO
NH2
50 kD
those IgG and Fab samples that
OPO32−
37 kD
HO
+
were first activated with PLP
Me
OPO32−
N
H
25 kD
(Figure 4, panel d). Nitrocellu+
Me
3: PLP (10 mM)
N
lose membranes displaying the
3:
+ −
+ −
+
− +
−
H
6:
+ +
+ +
+
+ +
+
Pyridoxamine
FLAG epitope were used to capO
T (°C): 37 37 50 50 37 37 50
50
H
N Protein
ture the antibodies through subProtein:
1
1
1
1
2
2
2
2
R
strate binding. The blots were
O 4
c
12% gel
10% gel
12% gel
then probed with ␣-biotin mAbs
R′ONH2
75
kD
conjugated to HRP to confirm the
SO3−
SO3−
+
R′O
presence of the biotin group.
N
H2N
50 kD
NH2
O
H
N Protein
Chemiluminescent output was
R
37 kD
observed only for samples that
O
5
HO2C
had been treated with PLP prior
H
H
25 kD
N
to biotinylation (Figure 4,
N
ONH2
5
panel e, bottom panel). Thus,
O
O
20 kD
DTT: + + +
+
− − − −
+
+
6: AlexaFluor 488 alkoxyamine
the presence of a signal for bioti3: + − +
−
+ − + −
+
−
nylated IgG and Fab, in corrobo7: + + +
+
+ + + +
+
+
O
n
MeO
ONH2
T (°C): 37 37 50 50 37 37 50 50
50 50
ration with the ELISA data, demProtein: 1 1
1
1
2
2
2
2
2
2
7: PEG-alkoxyamine (MW = 2000)
onstrates that these modified
samples retain their ability to
Figure 2. Regiospecific modification of antibodies. a) Reaction scheme for the modification of 1 and 2 by PLP
recognize and bind the FLAG
(3). b) SDS-PAGE analysis after fluorescent labeling of PLP-modified 1 and 2 with AlexaFluor 488 alkoxyepitope after the PLP reaction
amine (6). All samples were reduced with DTT before analysis, which separates the individual peptide
and subsequent oxime formachains. c) Samples of 1 and 2 were treated with PEG-alkoxyamine 7 after incubation with 3. SDS-PAGE
analysis revealed the presence of PEG conjugates (indicated by the red arrows) only after PLP treatment. For
tion. For other conjugates, we
1, modification of the light chain is readily observed under reducing (ⴙDTT) conditions (25% at 37 °C and
anticipate that the influence on
47% at 50 °C). Much lower levels of modification were observed for the heavy chain, preventing quantitaantigen binding will depend
tion. For 2, a single modification (20% at 37 °C and 42% at 50 °C) is observed under nonreducing (–DTT)
largely on the identity of the
conditions. Under reducing conditions, the modified light chain cannot be resolved from the heavy chain.
alkoxyamine that is installed.
Having shown that antigen binding is re- some antibody and other protein substrates molecules, including DNA strands and
carbon nanotubes. Additional studies seek
that cannot tolerate the overnight incubatained after modification with PLP, we
tion steps at elevated temperatures, as this to explore the attachment of environwanted to confirm that this strategy could
mentally sensitive fluorophores that can
behavior is likely to vary on a protein-toalso be used to modify antibodies with
report antigen binding for sensing
protein basis. Nonetheless, our preliminary
other binding specificities. Using SDSPAGE analysis after treatment with 6, fluo- results suggest that this method will be suf- applications.
ficiently broad in scope to be applied to a
rescent labeling of two other mAb subMETHODS
variety of situations.
strates, mouse ␣-actin (9) and mouse
Preparation of Carbonyl-Containing mAbs and
This site-specific strategy thus provides
␣-biotin-peroxidase (10), was observed
Fab. A 600 ␮L Eppendorf tube was charged with
(Supplementary Figure 5). In addition, fluo- a mild and facile approach to access well- a solution of IgG (1 delivered as 100 ␮L of a
5 mg ml⫺1 solution in 10 mM sodium phosdefined antibody conjugates. Current efrescent conjugates were obtained for polyphate, 150 mM NaCl, pH 7.4, with 0.02% soforts in our lab are utilizing this methodol- dium azide; 1 equiv) and a solution of PLP
clonal goat ␣-mouse antibodies (11). Unogy to attach antibodies to other functional (3 delivered as 100 ␮L of a 20 mM solution in
doubtedly, we expect that there will be
www.acschemicalbiology.org
VOL.2 NO.4 • 247–251 • 2007
249
H
O
HO
O
Mass, amu
49,000
3515
3058
3338
3041
3077
3095
2804 2824
2915
2783
3514
3337
3042 3058
3077
3094
2783
2803
2824
2913
2499
2542
2114 2109
4000
Mass, m/z
4000
1500
3041
3077
3095
3166
3283
3338 3328
2825
2909
2978
2544
2604
2427
2804
3058
2109
2115
1515
MALDI MS, intensity
(viii)
2122
2222 2176
2282 2297
2317
2409 2427
1988
2091
1816
1515
MALDI MS, intensity
48,492
(vii)
R
O
Mass, m/z
1500
f
(iv)
(v)
(vi)
O
s s
Modified Fab
MW = 47,908 (light chain modified)
MW = 47,954 (heavy chain modified)
MW = 48,015 (both chains modified)
2544
2122 2114 2109
2222 2176
2281
2315
2410
2427
MALDI MS, intensity
48,265
48,298
49,000
48,490
47,407
(iv) 5 (light chain only)
(v) 5 (heavy chain only)
(vi) 5 (both chains)
(vii) 5 (light chain) + PLP
(viii) 5 (both chains) + PLP
47,908 47,849
Mass, amu
47,204
O
1500
(iii)
48,222
48,266
48,298
47,848
47,803
49,000
e
47,932
47,976
48,051
47,406
47,206
ESI MS, intensity
(ii)
c
ESI MS, intensity
48,104
Mass, amu
48,032
47,000
b
1515
47,848
47,976
(i) Unmodified Fab (2)
N
H
N
5
d
47,954
48,014
48,034
48,156
48,264
ESI MS, intensity
(i)
47,000
MW = 123
s s
Decarboxylated Fab
MW = 47,803
a
47,000
H
N
4
s s
Intact Fab
MW = 47,848
(ii) Fab − 45 amu (4)
(iii) 4 + PLP
N
R
O
2
O
ONH2
O
H
N
3670
H
N
3671
MW = 247
O
O
Mass, m/z
3670
NH2
N
H
2794
O
Me
2092
2121
2177
2221
2281
2315
2411
H 2N
R
H
N
1815
H
N
1738
HO
OPO32−
+
3514
O
4000
Figure 3. MS analysis of Fab modification. Electrospray ionization (ESI) MS spectra for a) unmodified Fab, b) Fab after exposure to PLP, and c) PLPactivated Fab further elaborated with benzylalkoxyamine. The new reaction products, along with their expected masses, are indicated pictorially
above the spectra. In addition to the expected reaction products, a PLP adduct (ⴙ247) can be observed in panels b) and c). The loss of mass after
PLP activation corresponds to a decarboxylation of the N-terminal aspartate residue. MALDI MS spectra for proteolytic digest of d) unmodified
Fab, e) Fab after exposure to PLP, and f) PLP-activated Fab further elaborated with benzylalkoxyamine. The red box denotes the region in which
the N-terminal fragment is observed. As for the intact Fab, decarboxylation e) followed by oxime formation f) is observed, though no PLP adducts are
found.
25 mM phosphate buffer, pH adjusted to 6.5
with 1 M NaOH; 600 equiv). The mixture was
briefly agitated to ensure proper mixing and was
incubated without further agitation at 37 or 50 °C
for 18 –20 h. The PLP was removed from the reaction mixture via ultracentrifugation, exchanging
into 10 mM phosphate buffer, pH 6.5, with
0.02% sodium azide.
Oxime Formation. Carbonyl-containing samples
of IgG and Fab were prepared by the procedure described above. An aliquot of the purified mixture
(20 ␮L, ⬃2 mg mL⫺1 overall) was treated with the
alkoxyamine of interest (final concentrations of
0.21–250 mM, depending on the alkoxyamine).
This mixture was briefly agitated to ensure proper
mixing, and was incubated at RT for 18 –20 h. For
more specific details regarding oxime formation,
please see the Supporting Information.
Acknowledgments: The authors thank the U.S.
Department of Energy Nanoscale Science and Engineering Technology program for financial support, as
well as the Department of Chemistry at the University of California, Berkeley. We gratefully acknowl-
250
VOL.2 NO.4 • 247–251 • 2007
edge T. Iavarone and the QB3/Chemistry Mass
Spectrometry Facility for help with ESI analysis
(National Institutes of Health grant number
1S10RR022393-01). Additionally, we thank A. Falick
and the Howard Hughes Medical Institute facility for
help with MS/MS analysis. We would also like to
thank the Bertozzi group, J. Antos, E. Kovacs, and A.
Presley for many helpful discussions.
Supporting Information Available: This material
is available free of charge via the Internet.
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LETTER
a
b
1.5
0.5
1.3
0.9
0.3
0.7
0.2
0.5
1.0
0.8
0.6
0.2
0.1
3: +
− + − N/A N/A
T (°C): 37 37 50 50
4 N/A
Protein: 1
1 N/A
1
1
1
Conc. (µg/mL): 100 100 100 100 100 N/A
+
−
+
−
0
N/A
4
37 37 50 50
2
2
2
2
2
200 200 200 200 200
N/A
N/A
N/A
N/A
c
0
d
Fab (2) samples
Background
0.5
500
5000
50,000
Concentration of 1 (ng/mL)
e
Western blot
Top: chemiluminescence
Bottom: coomassie
0.6
100,000
Dot blot
(chemiluminescence)
O
0.4
HN
H
0.3
1
NH
H
H
N
0.2
S
O
0.1
N
H
8: biotin alkoxyamine
+
+
−
+
50
2
2
50
2
2
N/A
N/A
N/A
N/A
N/A
2
O
ONH2
α-Mouse
peroxidase
10−20% gel
Absorbance
1 after activation with PLP
0.4
0.1
0.3
0
3:
8:
T (°C):
Protein:
Conc. (µg/mL):
Unmodified 1
1.2
Absorbance
Absorbance
Fab (2) samples
Background
0.4
1.1
1.4
IgG (1) samples
3:
8:
T (°C):
Protein:
α-Biotin
peroxidase
+
+
−
+
+
+
−
+
+
+
−
+
+
+
−
+
50
1
50
1
50
2
50
2
50
1
50
1
50
2
50
2
Figure 4. Retention of antigen binding ability after modification with PLP. a) Sandwich ELISA data for IgG (1) and Fab (2) shows little-to-no erosion
of signal after modification. b) For samples of 1 incubated with and without PLP at 50 °C, similar ELISA signals were obtained at multiple concentrations. c) ELISA data for samples of 2 (with and without PLP treatment) after biotinylation with 8 (18% conversion). Minimal change in signal indicates no change in binding ability under the conditions of oxime formation with 8. Biotin conversion is based on HABA quantitation. d) Western blot
analysis of PLP-modified antibodies after biotinylation with 8. Additional bands in the chemiluminescent image (lane 2) are due to impurities in the
protein starting material that are below the detection limit of Coomassie staining but are observed by Western Blot. e) Subsequent dot-blot analysis was carried out on nitrocellulose displaying the FLAG epitope (orange). After exposure to biotinylated 1 and 2, the membranes were probed with
(i) an ␣-mouse-peroxidase conjugate to detect the presence of the antibodies and (ii) an ␣-biotin-peroxidase conjugate to confirm that they displayed the biotin group (shown schematically above the blot images).
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