Site-specific chemical modification of recombinant
proteins produced in mammalian cells by using
the genetically encoded aldehyde tag
Peng Wua, Wenqing Shuia, Brian L. Carlsona, Nancy Hua, David Rabukaa, Julia Leea, and Carolyn R. Bertozzia,b,c,d,1
Departments ofaChemistry andbMolecular and Cell Biology andcHoward Hughes Medical Institute, University of California, Berkeley, CA 94720-1460;
anddMolecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA 94720-1460
Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved December 3, 2008 (received for review August 7, 2008)
The properties of therapeutic proteins can be enhanced by chem-
ical modification. Methods for site-specific protein conjugation are
critical to such efforts. Here, we demonstrate that recombinant
proteins expressed in mammalian cells can be site-specifically
modified by using a genetically encoded aldehyde tag. We intro-
duced the peptide sequence recognized by the endoplasmic retic-
ulum (ER)-resident formylglycine generating enzyme (FGE), which
can be as short as 6 residues, into heterologous proteins expressed
FGE produced products bearing a unique aldehyde group. Proteins
bearing this ‘‘aldehyde tag’’ were chemically modified by selective
reaction with hydrazide- or aminooxy-functionalized reagents. We
applied the technique to site-specific modification of monoclonal
as membrane-associated and cytosolic proteins expressed in mam-
antibody engineering ? bioorthogonal reaction
Examples include growth factors, hormones, cytokines, replace-
ment enzymes, clotting factors, and monoclonal antibodies, the
fastest growing class of protein drugs (2). In many cases, the
intrinsic properties of therapeutic proteins can be improved by
attachment of polyethylene glycol (PEG) chains, a process also
termed PEGylation, which can improve pharmacokinetic prop-
erties (4). Chemical modification can also expand a protein’s
capabilities, such as conversion of an antibody into a drug or
diagnostic targeting element (5).
The major technical challenge in protein modification is
achieving site selectivity for the production of a homogenous
product. Most protein modification methods exploit the reac-
tivities of endogenous functionality such as the amino group of
lysine and the thiol group of cysteine (6). However, because
proteins often possess multiple copies of these residues, site-
specific labeling can be difficult to achieve. Thus, several groups
are chemically orthogonal to the 20 proteogenic amino acid side
chains. One approach involves engineering unique combinations
of natural residues such as the tetra cysteine motif that reacts
with biarsenical probes (7). Alternatively, a unique functional
group that is not found in natural amino acids can be installed
at the intended modification site and then reacted in a second
step with the moiety of interest. Ketones and azides epitomize
the kinds of functional groups that have been exploited for this
purpose (8). These electrophiles can be site-specifically incor-
porated into proteins by using a variety of elegant methods,
including chemical modification of a protein’s N terminus (9),
unnatural amino acid mutagenesis (10), or through the use of
enzymes that transfer prosthetic groups to the protein (11–13).
These methods are powerful research tools, but practical issues,
ecombinant proteins are now among the portfolio of clinical
candidates in most major pharmaceutical companies (1).
such as scalability and generality across proteins and expression
We recently reported a method for redirecting endogenous
cellular machinery to introduce aldehydes into recombinant
proteins. The method exploits formylglycine-generating enzyme
(FGE) (14), which converts cysteine to formylglycine (FGly)
1 sulfatases (Fig. 1A) (15). The modification is thought to occur
cotranslationally, before protein folding, and is critical for the
sulfatases’ catalytic function. We found that the consensus
sequence can be installed within heterologous proteins ex-
pressed in Escherichia coli, where it is modified efficiently by a
coexpressed bacterial FGE (16). Furthermore, the minimized
6-residue sequence LCxPxR, derived from the most highly
conserved portion of the FGE recognition site, also directed
efficient conversion of cysteine to FGly. The isolated proteins
were thus outfitted with an aldehyde group for site-specific
chemical modification with aminooxy- or hydrazide-functional-
ized moieties, including fluorophores, affinity tags, and PEG
chains (Fig. 1B). We demonstrated the generality of the tech-
nique, which we call the ‘‘aldehyde tag,’’ in prokaryotic expres-
sion systems by modifying several proteins at different locations
within their primary sequence.
A vast majority of pharmaceutically relevant proteins, how-
ever, must be expressed in mammalian cells due to their re-
quirement of various posttranslational modifications (17). Di-
sulfide bonds, glycosylation, and sulfation, for example, are
difficult or impossible to achieve in bacteria or yeast. Thus, we
sought to extend the aldehyde tag technology to proteins ex-
pressed in mammalian cells. Conveniently, in eukaryotes, FGE
is located in the endoplasmic reticulum (ER) where it modifies
sulfatases destined for lysosomes or secretion (14). We proposed
that heterologous proteins endowed with an aldehyde tag se-
quence might also be modified by mammalian FGE as they
traverse the secretory pathway.
Here, we demonstrate that recombinant proteins expressed in
mammalian cells can be site-specifically modified by using the
aldehyde tag technology. We initially focused on monoclonal
antibodies because of their clinical importance. The most widely
used isotype, IgG, has a conserved N-linked glycosylation site in
the Fc region of each heavy chain that is important for protein
structure and for various effector functions (18). Because of this
posttranslational modification as well as several disulfide bonds,
Author contributions: P.W. designed research; P.W., W.S., B.L.C., N.H., D.R., and J.L. per-
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2009 by The National Academy of Sciences of the USA
March 3, 2009 ?
vol. 106 ?
functional IgGs can only be produced in mammalian (or insect)
expression systems. Conventional methods for IgG labeling
involve nonspecific coupling reactions with lysine residues on the
protein surface. Because lysines may lie near the antigen-binding
site of some antibodies, there is a risk that these labeling
strategies will reduce antibody function. The aldehyde tag would
offer a more controlled method for covalent modification, one
that could potentially be tailored for any chosen site on the
Accordingly, we engineered both Fc and intact IgG constructs
bearing aldehyde tags at various sites. When expressed in CHO
or HEK cells, the proteins were efficiently modified with alde-
hyde groups that were exploited for site-specific modifications.
Furthermore, we demonstrated that membrane-associated pro-
teins can be labeled with aldehyde tags and then chemically
expressed in HEK cells can be modified by a coexpressed
prokaryotic FGE directed to that compartment. The aldehyde
tag is therefore a robust and general method for modification of
proteins produced in mammalian systems.
Results and Discussion
Implementation of the aldehyde tag in mammalian systems was
first explored by using the IgG Fc fragment as a model protein.
cells lines, affording the quantities needed for detailed chemical
analysis. In addition, the Fc domain has therapeutic relevance on
its own (19) and as a fusion with other proteins (20). Using the
commercial pFuse vector (InvivoGen), we generated Fc con-
structs encoding either a 13-residue aldehyde tag sequence
derived from human arylsulfatase A (LCTPSRAALLTGR,
‘‘Ald13’’) or the minimized 6-residue tag LCTPSR (‘‘Ald6’’)
situated at either the N or C terminus (4 constructs in total). As
controls, we also generated constructs in which the cysteine
residue modified by FGE was mutated to an alanine residue.
The constructs were introduced into Chinese hamster ovary
(CHO) cells by transient transfection in serum-free medium and
the expressed proteins were isolated by using protein A/G (a
genetically engineered protein that contains Fc-binding domains
of both protein A and protein G) agarose. All 4 proteins
expressed at approximately the same levels, which were compa-
rable with the expression level of the unmodified Fc fragment
(?1.0 mg/L). To probe for the presence of aldehydes, the
proteins were reacted with an aminooxy-FLAG peptide (DYK-
DDDDK) conjugate (16) (Fig. 2A illustrates a C-terminally
modified Fc fragment) at pH 5.5 and analyzed by 4–12%
SDS/PAGE and Western blot. The Fc proteins bearing Ald13or
Ald6 tags at either the N or C terminus all showed robust
labeling, whereas the control C to A proteins gave no detectable
signal (Fig. 2B; only Fc proteins with C-terminal Ald13 and
N-terminal Ald6are shown; the other 2 proteins gave similar
results). Tryptic digestion of the aldehyde-tagged Fc proteins
allowed direct identification of FGly by mass spectrometry (Fig.
2C). From all 4 proteins, the FGly-containing tryptic peptide as
well as the unconverted cysteine-containing peptide were readily
To determine the extent of conversion of Cys to FGly for all
of the Fc constructs, we doped an isotope-labeled peptide into
the digestion products from individual Fc constructs as an
internal standard for mass spectrometry quantitation (21). The
internal standard possessed the same sequence as the Cys-
containing peptide, thus allowing direct determination of its
concentration within each Fc protein digest. By subtraction, the
fraction of FGly-containing peptide could be calculated to
aldehyde tag. (A) FGE oxidizes a critical cystein to FGly within a conserved
13-amino acid sequence. (B) The aldehyde tag can be transported into a
heterologous protein for site-specific modification with chemical probes.
Site-pecific protein modification using the genetically encoded
to the gene of IgG Fc fragment. Upon expression, the encoded cysteine is
modified to an aldehyde, which can be used as a chemical handle for reaction
with an aminooxy FLAG probe. (B) (Upper) Western blot probed with ?-FLAG
antibody. (Lower) Coomassie blue-stained image of the gel. (C) Mass spectra
(Left) Mass spectrum of the tryptic fragment incorporating FGly. Theoretical:
incorporating unmodified Cys after treatment with 2-iodoacetamide. Theoreti-
cal: 546.2721 m/z, observed: 546.2811.
Site-specific labeling of aldehyde-tagged IgG Fc domain. (A) Nucle-
Wu et al.
March 3, 2009 ?
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no. 9 ?
determine the conversion efficiency. The overall conversion
efficiency ranged from 25% to 67% for the various constructs
tested (Table 1).
The relatively low conversion efficiencies for some of the
constructs might reflect an inadequate supply of endogenous
FGE for complete modification of the overexpressed Fc proteins
as they mature in the ER. In prokaryotic systems, we found that
co-overexpression of a heterologous FGE can increase the
conversion efficiency of a tagged protein (16). Accordingly, we
overexpressed human FGE (hFGE) in CHO cells, either tran-
siently or stably, along with transient expression of the Fc
constructs. A shown in Table 1, hFGE coexpression increased
the conversion efficiency for each Fc construct. In most cases,
the most dramatic improvements were observed with transiently
overexpressed hFGE. For a list of the oligonucleotides used in
this study, see Table S1.
The selectivity of the aldehyde-aminooxy condensation reac-
tion was demonstrated by selective modification of aldehyde-
tagged Fc protein in the context of crude conditioned medium.
CHO cells were transiently transfected with N-Ald13-Fc along-
side hFGE. The conditioned medium was collected and dialyzed
against PBS to remove competing metabolites (e.g., pyruvate,
which has a reactive ketone, and glucose, which possesses an
aldehyde) while retaining proteins. The dialyzed medium was
reacted with aminooxy-FLAG and analyzed by Western blot. As
shown in Fig. 3, the Fc protein was the only species detected,
despite the presence of other abundant proteins in the sample.
Notably, the only endogenous cellular proteins predicted to
possess FGly residues are the lysosomal and secreted sulfatases
as well as a few kinesin family member proteins (Table S2), none
of which were visible in the CHO cell conditioned medium. In
model studies, we observed no covalent labeling of commercial
sulfatases with hydrazide probes, presumably because their FGly
residues are buried in the active site of the folded protein (data
not shown) (15). Thus, among a complex mixture of cellular
proteins that includes active sulfatases, aldehyde-tagged heter-
ologous proteins may be the only substrates that label with
aminooxy- and hydrazide-functionalized reagents.
Modification of an Aldehyde-Tagged Full-Length IgG. We extended
the aldehyde tag method to site-specific modification of a
full-length monoclonal IgG specific for Nogo receptor-2 (NgR2)
(22). This antibody comprises Fab fragments derived from phage
display, fused to a human Fc domain by using the pIgG construct
(23). We cloned the 6-mer aldehyde tag sequence (LCTPSR)
into the C terminus of the CH3 region of the heavy chain. This
protein and the light chain, encoded in the same plasmid, were
expressed transiently in HEK293T suspension cells alongside
hFGE (2:1 wt/wt). The protein was purified from the condi-
tioned medium by using protein A-agarose, then reacted with
aminooxy-biotin and analyzed by SDS/PAGE and Western blot
(Fig. 4A). The intact antibody was specifically labeled, and
location of the modified aldehyde tag on the heavy chain (Fig.
S2–S3). As before, a C108A mutation in the aldehyde tag
sequence abrogated labeling. In addition, we conjugated the
gen) and investigated the ability of the labeled antibody to bind
medium. (A) Coomassie blue-stained image of the gel. (B) Western blot
probed with ?-FLAG antibody.
Site-specific labeling of N-Ald13-Fc domain in crude conditioned
Table 1. Percent conversion of Cys to FGly in recombinant Fc fragments
Tag location Tag typeCHO cells
CHO cells transiently
cotransfected with hFGE¶
CHO cells stably
transfected with hFGE?
40 ? 8
67 ? 1
28 ? 1
45 ? 2
44 ? 2
91 ? 2
45 ? 1
69 ? 3
68 ? 12
77 ? 3
62 ? 3
64 ? 2
The standard deviation represents the error of at least 3 replicate experiments.
*The tag was inserted downstream of the signal peptide sequence.
†The tag comprised the C-terminal residues of the protein. Two residues upstream of the tag sequence were
mutated in order to generate a Kpnl restriction site.
‡The 6-mer sequence was LCTPSR.
§The 13-mer sequence was LCTPSRAALLTGR.
¶Human FGE (hFGE) was encoded on a separate plasmid, pcDNA3.1.
?The procedure used for generating the stably transfected CHO cell line is in SI Text.
labeling of the monoclonal ?-Nogo R2 IgG with aminooxy biotin probe.
(Upper) Western blot probed with ?-biotin antibody. (Lower) Coomassie
blue-stained image of the gel. (B) Fluorophore conjugated ?-Nogo R2 IgG
retains binding specificity.
Labeling of the aldehyde-tagged full-length IgG. (A) Site-specific
www.pnas.org?cgi?doi?10.1073?pnas.0807820106 Wu et al.
recombinant Nogo R2 expressed on HEK293 cells (Fig. 4B).
Importantly, the modified antibody retained its antigen binding
activity. In a control experiment, only background signals were
detected when HEK293 cells expressing Nogo R1 were treated
with the labeled antibody and analyzed by flow cytometry (data
Labeling of Aldehyde-Tagged Cell Surface Proteins. Like secreted
proteins, plasma membrane-associated proteins traffic through
the secretory pathway and are therefore potential substrates for
ER-resident FGE. To test this notion, we introduced the 13-mer
aldehyde tag at the N terminus of the platelet-derived growth
factor receptor (PDGFR) transmembrane (TM) domain en-
coded in the pDisplay vector (Invitrogen). The construct was
transiently expressed in CHO cells along with hFGE. After 48 h,
the cells were reacted with biotin hydrazide then stained with
Alex Fluor 488-streptavidin and analyzed by flow cytometry
(Fig. 5A). As shown in Fig. 5A, CHO cells expressing aldehyde-
tagged PDGFR-TM (Ald13-TM) showed a marked increase in
fluorescence compared with cells expressing the unmodified
PDGFR-TM protein or the C18A mutant. These results were
mirrored by using fluorescence microscopy (Fig. 5B). We were
not able to perform a detailed quantitation of Cys to FGly
of this membrane-associated protein.
Similarly, we inserted an aldehyde tag at the N terminus of
recombinant mouse CD4, a membrane-associated protein in-
volved in T cell activation (24). As demonstrated by using
fluorescence microscopy, CHO cells expressing aldehyde-tagged
Fluor 488, whereas cells expressing the corresponding C18A
Labeling of an Aldehyde-Tagged Cytosolic Protein. We were curious
whether cytosolic proteins in mammalian cells could be labeled
by using the aldehyde tag method. Endogenous FGE is report-
edly confined to the ER, where it is glycosylated and possibly
associated with other ER-resident proteins (14). Thus, for
soluble cytosolic expression, we cloned a bacterial FGE homolog
derived from Streptomyces coelicolor (25) into a mammalian
expression vector. The GFP derived from Aequorea coerulescens
was modified with the 13-mer aldehyde tag downstream of an
N-terminal His6 tag. This construct, called Ald13-GFP, was
transiently expressed in HEK293T cells along with the S. coeli-
color FGE. After cell lysis and nondenaturing Ni-NTA purifi-
cation, the protein was reacted with biotin-hydrazide and ana-
lyzed by nonreducing PAGE and Western blot.
As shown in Fig. 6, the aldehyde-tagged GFP, but not the
C10A mutant, was effectively labeled with biotin hydrazide.
Labeling depended on coexpression of S. coelicolor FGE. How-
ever, GFP expressed in the absence of S. coelicolor FGE
appeared to undergo conversion of Cys to FGly at a low level.
The Coomassie-stained gel of GFP expressed together with S.
coelicolor FGE revealed 2 bands, one migrating at 32 kDa
corresponding to the monomeric protein and another at 64 kDa
corresponding to the disulfide-bound dimer. The ratio of these
2 species reflects the percent conversion of Cys to FGly.
Accordingly, the C10A mutant appeared as a single band at 32
kDa. When GFP was expressed without S. coelicolor FGE, a
majority of the protein migrated at 64 kDa, but a minor amount
migrated with an apparent molecular mass of 32 kDa, consistent
with a low level of Cys modification.
Taken together, the PAGE and Western blot data suggest that
aldehyde-tagged GFP expressed in the cytosol of HEK293 cells
undergoes Cys to FGly conversion at low levels in the absence of
exogenously expressed cytosolic FGE. We considered the pos-
sibility that aldehyde-tagged GFP was exposed to endogenous
FGE during the process of cell lysis, despite our efforts to
prevent disruption of the ER membrane by use of nondetergent
buffers. To test this, we analyzed the isolated cytosolic fraction
for the presence of BiP, a major ER-resident chaperone. West-
ern blots confirmed that this protein was present at low levels in
the cytosolic fraction, consistent with a small degree ER con-
tamination (Fig. S4). Alternatively, there may exist a previously
cytometry analysis of CHO cells transfected with Ald13-TM or unmodified
PDGFR-TM (TM) or Ald13-TM (C18A). At 48 h after transfection, cells were
labeled with biotin hydrazide [1 mM in PBS (1% FBS) (pH 6.5) for 1 h] and
stained with Alex Fluor 488-labeled streptavidin. Error bars represent the
standard deviation of the mean for 3 replicate ligation reactions. Similar
results were obtained in 3 replicate experiments. MFI, mean fluorescence
intensity; AU, arbitrary units. (B) Fluorescence micrographs of CHO cells trans-
fected with Ald13-TM (Left) or TM (Right) and labeled with aminooxy Alexa
Fluor 647. (C) Fluorescence micrographs of CHO cells transfected with Ald13-
CD4 (Left) or Ald13-CD4(C18A) (Right) and labeled with Alexa Fluor 488. Dye
labeling reactions were performed at pH 6.4 for 1 h at room temperature.
33342 before imaging. Blue, DAPI channel; green, FITC channel; red, Cy5
Labeling of membrane-associated Ald13-TM and Ald13-CD4. (A) Flow
Ald13-GFP and Ald13-GFP (C10A) plasmids were transiently transfected into
HEK 293T cells with (?) or without (?) S. coelicolor FGE. Three days after
Ni-NTA agarose. (Upper) ?-Biotin Western blot of aldehyde-tagged GFP con-
structs after reaction with biotin hydrazide. (Lower) image of the Coomassie-
Labeling of Ald13-GFP after cytosolic expression in HEK cells. The
Wu et al.
March 3, 2009 ?
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no. 9 ?
uncharacterized FGE activity in the cytosol of human cells.
Likewise, FGE-like activities have been identified in E. coli
and postulated in Caenorhabditis elegans, but the molecular
identities of the corresponding enzymes have not yet been
The aldehyde tag offers a practical and versatile method for
site-specific chemical modification of secreted, membrane-
associated, and cytosolic proteins expressed in mammalian sys-
tems. The reactions of aldehydes with aminooxy or hydrazide
reagents are typically complete within 2 hours at 37 °C, and the
resulting oximes and hydrazones, respectively, are quite stable
under physiological condition. Moreover, many aminooxy- and
hydrazide-functionalized reagents are commercially available;
thus, the procedure requires only simple cloning steps to gen-
erate the necessary components for protein modification. With
minimal optimization, we obtained recombinant proteins with
?90% conversion of Cys to FGly. We expect that further
manipulation of FGE expression levels and exploration of
different cell lines will produce systems capable of even higher
conversion. Importantly, we discovered that both 6-mer and
13-mer tags are viable for protein modification applications,
although the 13-mer tag provides higher conversion efficiency of
Cys to FGly in the IgG construct studied. It is notable that the
FGE consensus sequence is highly restricted to Type I sulfatases,
wherein the reactive aldehydes are buried in the active site.
Analysis of the human genome sequence reveals very few
proteins outside of this family with related sequence motifs
(Table S2). Thus, even within unpurified cell lysates or condi-
tioned media, recombinant aldehyde-tagged proteins are pre-
dicted to be the predominant species that label with aminooxy or
The presence of an unmodified Cys residue within the alde-
hyde tag sequence has some interesting consequences, both
advantageous and potentially disadvantageous. In some cases,
we found that the unmodified proteins formed disulfide-bound
homodimers, which should enable their trivial separation from
the corresponding monomeric FGly-containing proteins by size-
exclusion methods. However, it is possible that unconverted Cys
residues will interfere with folding of proteins possessing native
disulfide bonds, although we observed no such detrimental
effects in the cases of Fc and IgG.
The proteins reported in this work were labeled near their N
or C termini. The natural substrates for FGE, Type I sulfatases,
possess internal consensus sequences, suggesting that internal
well. An interesting extension of this work will be to insert
aldehyde tag sequences into internal loops of recombinant
proteins, perhaps at sites normally occupied by glycosylation or
other posttranslational modifications. In this way, the aldehyde
tag could be exploited to install a wide range of natural or
unnatural posttranslational modifications anywhere on the sur-
face of a folded protein. The precise chemical control offered by
the aldehyde tag method should enable the development of new
protein products for research and therapeutic purposes.
Materials and Methods
Protein Modification Reactions. Chemical modification of aldehyde-tagged
proteins was achieved by treating 4 ?g of the target protein with 300 ?M
aminooxy- or hydrazide-functionalized probe [Alexa Fluor 488 C5-aminooxy-
acetamide (Invitrogen), biotin hydrazide (Sigma), or aminooxy-FLAG (16)] in
labeling buffer [100 ?M Mes (pH 5.5), 1% SDS] at room temperature for 2 h.
After adding 4? SDS/PAGE loading buffer to each sample, reaction mixtures
were resolved on Bis-Tris Criterion gels (4–12% or 12%; Bio-Rad). Protein
loading was determined by Coomassie staining. Western blots were probed
with either an ?-biotin-HRP antibody (1:100,000 dilution; Jackson Immuno-
Sigma) and developed by using SuperSignal West Pico Chemiluminescent
Substrate. Aminooxy-FLAG (H2NO-CH2-CO-NH–(DYKDDDDK)–CO2H) was syn-
thesized via standard Fmoc-based solid-phase peptide synthesis protocols as
previously described (27). The final residue added at the N terminus was
(t-Boc-aminooxy)acetic acid followed by cleavage under standard conditions.
The peptide was subsequently purified by C18reversed-phase HPLC.
Tryptic Digestion of Aldehyde-Tagged Fc. After affinity purification using
protein A/G agarose (protein A/G is an engineered construct possessing Fc
binding domains from both protein A and protein G), each aldehyde-tagged
Fc fragment was subjected to tryptic digestion. Each protein was first treated
with Tris(2-carboxyethyl) phosphine hydrochloride (TCEP, 5 mM) at 37 °C for
30 min, followed by alkylation using iodoacetamide (20 mM) at room tem-
(Promega) is added at 1:20 (mass ratio, enzyme/substrate) for overnight
digestion. The completeness of protein digestion was verified by silver stain.
Quantification of the Conversion Efficiency of Cysteine to Formylglycine in the
Aldehyde-Tagged Fc. Peptide standards were custom synthesized by Biomer
Technology. Heavy isotope-labeled peptides were used as the internal stan-
of the peptide standards for quantifying the N- and C-tagged Fc are ISLCTPSR
isotope-labeled counterpart, which increased the mass unit by 7 Da; whereas
both Leu and Gly residues in SLGTLCTPSR were isotope labeled, which in-
creased the peptide mass by 10 Da. The Cys of the peptide standards was
carboxymethylated to have the same modification as the Fc digests.
LC-ESI-MS (Waters Micromass Q-TOF) and MALDI-TOF/TOF (ABI 4800) were
used for quantifying the occupancy of the aldehyde tag on Fc. Individual Fc
digest from 160 fmol of protein was mixed with 50 fmol of its corresponding
internal standard, and the mixture was injected into LC-ESI-MS. MS/MS mode
was applied in some cases to acquire the peptide sequence data. For MALDI-
TOF/TOF analysis, both Fc digests and internal standards were diluted to 2
a MALDI sample plate, followed by loading of the matrix solution (?-cyano-
4-hydroxycinnamic acid in 60% of CH3CN, 0.1% TFA). The signal of both Fc
peptides and internal standards were within the linear range of detection by
a given mass spectrometer. The peak ratio of a pair of unlabeled peptide and
its labeled counterpart (of known quantity) shown in the mass spectrum
allowed us to calculate the absolute amount of the unconverted peptide
(unlabeled) derived from Fc. Finally the occupancy of the aldehyde tag on a
given Fc was deduced from the quantity of the unconverted peptide and the
total amount of Fc protein. An equation is shown below to illustrate the
calculation. Occupancy of the aldehyde tag on Fc ? 1 ? [(the quantity of
doped peptide standard ? the isotope ratio*)/the quantity of Fc protein
Labeling of Ald13-TM Expressed on CHO Cells and Flow Cytometry Analysis. CHO
ified PDGFR-TM and hFGE (2:1 wt/wt). After 40 h, the cells were washed,
collected, and resuspended in PBS containing 0.1% FBS (vol/vol) (pH 6.4) and
200-?L aliquots containing 5 ? 105cells were distributed into a V-bottom
96-well plate. Biotin hydrazide was added at 1 mM final concentration, and
the cells were incubated for 1 h at room temperature. Subsequently, the cells
were washed 3 times with PBS containing 1% FBS (vol/vol) (PBS/FBS) and
25 min, the cells were washed 3 times with 200 ?l of PBS/FBS and resuspended
in 400 ?l of PBS/FBS. Flow cytometry was performed by using a FACScalibur
instrument (Becton Dickinson).
Analysis of Ald6-Tagged Anti-Nogo R2 IgG Binding Activity. HEK293F cells were
transiently transfected with the human NgR2 in pCEP4 plasmid (Invitrogen)
in PBS/FBS, and 50-?L aliquots containing 5 ? 105cells were distributed into a
V-bottom 96-well plate. Alexa Fluor 488-conjugated Ald6-IgG (anti-Nogo R2)
was added at a final concentration of 6 ?g/mL, and the cells were incubated
(vol/vol) and resuspended in 400 ?L of the same buffer. All steps were carried
*Note that the isotope ratio refers to the peak ratio of the unconverted peptide vs. the
labeled peptide standard.
www.pnas.org?cgi?doi?10.1073?pnas.0807820106Wu et al.
out on ice. Flow cytometry was performed by using a FACScalibur instrument Download full-text
Fluorescent Microscopy Analysis of Labeled Membrane-Associated Proteins.
CHO cells were seeded at a density of 50,000 cells per milliliter into an 8-well
microscopy tray and allowed to grow for 24 h. The cells were then transiently
transfected with plasmids encoding Ald13-TM or Ald13-CD4, or with control
plasmids, unmodified PDGFR-TM or Ald13-CD4(C18A), along with hFGE (2:1
wt/wt). The cells were incubated for 48 h at 37 °C in a humidified 5% CO2
atmosphere in Ham’s F-12 medium supplemented with 10% FBS. The cells
were washed (2 times in 300 ?L of PBS) and incubated with aminooxy-Alexa
Fluor 647 (50 ?M) in PBS/FBS (pH 6.4) for 1 h. Hoechst 33342 dye (1 ?g/mL,
Invitrogen) was added, and after 1 min the cells were rinsed with PBS/FBS (pH
7.0, 3 times in 400 ?L) and resuspended in 400 ?L of the same buffer. The cells
were analyzed by fluorescence microscopy at 25 °C using a Zeiss Axiovert
1.4 N.A. PlanApochromat oil-immersion lens. Image stacks containing 40
sections, spaced 0.1 ?m apart, were acquired by using a CoolSNAP HQ CCD
camera (Photometrics). The image stacks were digitally deconvolved by using
the nearest-neighbor algorithm of Slidebook (Intelligent Imaging Innova-
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health Grants GM59907, 5PN2EY018241-02-NDC for the Optical Control of
Biological Function, and K99GM080585). The pIgG plasmid was provided by
Dr. Christoph Rader at the National Cancer Institute, Bethesda, MD.
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