Proc.Natl. Acad. Sci. USA
Vol. 87,pp.9848-9852, December 1990 Biochemistry
Molecular epitope identification by limited proteolysis of an
immobilized antigen-antibody complex and mass
spectrometric peptide mapping
(epitopemapping/molecularweightdetermination/plasmadesorption massspectrometry/complementcomponentC3a/ monoclonal antibody)
DETLEV SUCKAU*, JORG KOHLt, GABRIELE KARWATHt,
KLAUSSCHNEIDER*,
MONIKACASARETTOt,
DIETER
BITTER-SUERMANNt,
ANDMICHAEL
PRZYBYLSKI*§
*Fakultat furChemie,UniversitatKonstanz, D-7750 Konstanz, Federal Republic of Germany;tInstitut furMedizinischeMikrobiologie,Medizinische Hochschule Hannover, D-3000 Hannover, Federal Republic of Germany; andtDeutschesWollforschungsinstitutAachen,D-5100Aachen,
FederalRepublicofGermany
Communicatedby FredW. McLafferty, September 10, 1990 (received for review July 9, 1990) ABSTRACT Sequences of antigenic determinants were
identified by limited proteolysis of peptide antigens bound to an immobilized monoclonal antibody and direct molecular weight determination of the monoclonalantibody-bound peptide frag-ments by
252Cf
plasma desorption mass spectrometry. The epitope peptides to the monoclonal antibody h453[Burger,
R., Zilow, G., Bader, A., Friedlein, A. & Naser, W. (1988) J. Immunol. 141, 553-558] wereisolated from immobilizedanti-gen-antibody complexes by partial trypsin digestion. A syn-theticeicosapeptide comprised of the C-terminal sequence of the humancomplement componentpolypeptide
des-Arg77-C3a
as well as guinea pigdes-Arg78-C3a was used as an antigen. Conditions were developed under which trypsin specifically degraded theantigenswithoutinactivation of the immobilized antibody. After proteolysis, epitope peptides were dissociated from theantibody with 4 MMgCl2. The antigenic peptides were purified by HPLC and identified by
252Cf
plasmadesorption massspectro! retry. The epitope recognized by h453 resides on the C-terminaltryptic peptides of human(residues70-76)and guineapig (residues70-77)C3a.Asanestimationof accuracy this method is able to provide, trypsin digestion of immune complexes caused cleavage of theantigenwithin adistance of two amino acid residues upstream from theepitope.A variety of methods have been applied to the study of monoclonal antibody
(mAb)-antigen
interactions and the characterization of theirrespective
epitopes.
Twomajor
approaches that have beenwidely employed
forepitope
characterization arecompetitive
binding analysis using
syn-thetic peptides and
fine
specificity
studies withpanels
ofevolutionary
variant orrecombinantproteins
(1).Although
wellestablished, these methodshavemajor limitations;
e.g.,discontinuousor
conformationally
definedepitopes
maynotbe detectable by
using peptide
probes
(2). Site-directed mutagenesisexperimentsin epitopestudies could begreatly facilitated if some information abouttheputative epitope isavailablein advance. Adirectapproach ofepitope mapping, which seems promising in this respect, has been more
re-cently introduced basedonthe
finding
that(i) mAbsexhibit remarkable resistance towardsproteolytic
enzymes,(ii)
inimmunecomplexes,
antigenic
determinantscanbeprotectedfromproteolytic degradation, and
(iii)
proteolysis does not lead to dissociation of immune complexes (3-6). Limitedproteolytic cleavage ofimmunecomplexeshas been usedfor epitopecharacterizationbymeansofPAGE(7)and
by
HPLC(6)of the respective
peptide digests.
However, HPLCsep-aration of complex digest mixtures followed by amino acid analysisorpeptide sequencingmay not enableunambiguous epitope identification duetounresolvedpeptides.
Thefeasibility of fastatom bombardment mass spectrom-etry(FABMS) and 252Cf plasma desorptionmassspectrometry
(PDMS) foraccuratemolecularweight determinationof
poly-peptides has been established in several bioanalytical appli-cations (8).Particularly, abundantmolecularions of polypep-tides up to small proteins have been obtained with high sensitivity by PDMS(9). Apromisingapproach inrecent work
hasbeentheapplicationtomulticomponentpeptidemixtures,
suchasproteolytic digests (peptide mapping; ref.8). Thehigh molecularspecificity provided bymassspectrometricpeptide mapping has been successfully used in protein structural studies, such as the characterization of cDNA-derived se-quences,identification ofposttranslationalmodifications, and differentiation of isoenzymestructures (10-12).
Inthisstudy the combination of limited enzymatic
prote-olysis
and PDMS has beenapplied to themolecularepitope analysis of complementcomponentC3a,which isrecognizedas a potent mediator of inflammation (13). Amino acid
sequences(14, 15) andtertiarystructure(16, 17)of C3a from severalspecies including human (77-amino acid residues) and guinea pig (78
residues)
have been reported. The three-residue C-terminal sequenceof C3arepresentstheessential receptor-binding site (18, 19). However, biologicalactivity
islostby C-terminal desargination (13), concomitant with the exposureofaneoantigenic determinant that is
recognized
by
mAb h453 (20). h453 coupled to
tresyl-activated
Sepharose
(h453-TAS) was used for the preparation of immunecom-plexes with (i)aneicosapeptide comprising human(h) C-ter-minalC3a, and (ii) guinea
pig
(gp)des-Arg78-C3a
(des-Arg78-gpC3a). For bothantigens,
massspectrometric
peptide
map-ping of tryptic peptides dissociated from the truncated
immunecomplex byMgCl2 directly establishedtheepitope,
whereas the nonepitopepeptides were identified after
sepa-ration fromtheimmunecomplex,as
schematically
illustratedinFig.1.Moreover, thesynthetic peptide (hC21)
comprising
the sequence[Gln
65,Arg66]hC3a-(57-77)
wasdesigned
topro-videanestimation ofthestericrequirements forthe ternary
trypsin-antigen-antibody complex, by
introducing
threeequidistant tryptic cleavage sites.
Abbreviations: E;S, enzyme-to-substrate ratio (wt/wt); gpC3a, guinea pigC3a; hC3a, human C3a;h453-TAS,mAbh453coupledto
tresyl-activated Sepharose; hC21, [Gln65,Arg66]hC3a-(57-77); hC21dR,des-Arg21-hC21; mAb,monoclonalantibody;PDMS,252Cf plasmadesorptionmassspectrometry;FABMS,fastatom bombard-mentmass spectrometry.
§Towhomreprintrequestsshould be addressed. 9848
Thepublicationcostsofthis articleweredefrayedinpartbypagecharge payment.Thisarticle musttherefore beherebymarked"advertisement" in accordance with18U.S.C. §1734solelytoindicatethisfact.
Proc. Nati. Acad. Sci. USA 87 (1990) 9849 Immune-Complex
I
0.2 ml of TS. Pooled supernatant and remaining immune complexes after dissociation were subjected to HPLC
puri-fication on a0.4 x 25 cm Nucleosil 300-7-C18 column
(Ma-cherey & Nagel). Elution was carried out with a linear
gradient of 0-52% acetonitrile in water containing 0.04% trifluoroacetic acid over 25 minat aflow rate of 1 ml/min.
Peptide fractions were detected with an M490 multiwave-length detector (Waters),lyophilized, andredissolvedin 5
,ul
of 0.1% trifluoroacetic acid for mass spectrometric analysis. Isolation of Epitope Peptides fromdes-Arg78-gpC3a. Sixty microgramsofdes-Arg78-gpC3awasallowedto bind to 500,g of immobilized h453 for1 hr at20'C.Thegel was rinsed withTS, and samples equivalentto6.4
gg
of bound antigenwere incubatedfor 30minat370C with 50gl
of TScontaining 0,5,or 45
/ig
of trypsin, After removal of supernatant and treatmentthreetimes with 1 ml ofTS, dissociation and HPLC analysis were performed as describedabove.Mass Spectrometry. Nitrocellulose surfaces for sample adsorptioninPDMS werepreparedasdescribed (24). Peptide
solutionswereallowedtoadsorb for 2-3minfollowedbyspin
drying (25). Spectra wereobtained on atime-of-flight spec-trometer(Bio-Ion 20 K,Uppsala, Sweden) at a 15-kV accel-erating voltage. FABMS was performed on a Finnigan (San
Jose, CA) MAT-312/AMD-5000 double-focusing spectrom-eter, with a 20-kV cesiumprimaryion source(AMD,
Beck-eln, F.R.G.); glycerol was used as a matrix for the sample.
RESULTS AND
DISCUSSION
FIG. 1. Scheme of the mass spectrometric epitope mapping method ofan antigen-antibody complex as compared to peptide mapping of the free antigen. The use ofan immobilized antibody
(mAb) allows the separation ofnonepitope and epitope peptides after limited proteolysis of the immune complex. Molecular ions of nonepitope and epitope proteolytic peptidesareillustrated by solid
and openbars, respectively.
MATERIALS AND METHODS
Preparation of Antigens and Immobilized Antibody. The peptide hC21, which has the sequence
[Gln65,Arg661hC3a-(57-77)was prepared by solid-phase synthesis (21) and
con-tained a Cys'-S-acetamidomethyl protecting group. gpC3a wasisolated and purifiedasdescribed (22). The desarginated
forms,des-Arg21-hC21 (hC2ldR) and des-Arg78-gpC3a,were
prepared by carboxy-peptidase B treatment (23) and were
purified by HPLC, which yielded -95% purity for
des-Arg-C3a. mAb h453 (20) was affinity purified using protein A-Sepharose (Pharmacia) and was coupled to tresyl-activatedSepharose (Pharmacia) accordingtothe supplier's procedures. The immobilized mAb (h453-TAS)wasstored in
phosphate-buffered salinecontaining 0.03% sodium azideat
a concentration of 800 ,ug of bound mAb per ml of gel
suspension.
Preparation and Dissociation of ImmuneComplexes. h453-TAS(1 ml)wasequilibrated with TS buffer (50 mM TrisHCI,
pH 7.5/150 mM NaCI), and 50 ,g of hC21dR in 0.5 ml of TS
was allowed to bind for 2 hr at 20°C. Free antigen was
removed by three washes each with 1 ml of TS, and the gel volumewas adjustedto 1 ml. Aliquots wereused either for
proteolysisorfordirect dissociationby addition of 0.4 ml of
4M MgCl2. Dissociation wasallowedtoproceed for 30min at37°C. The gel wasthen washedtwice with 0.15 ml of 4 M
MgCI2, and thepooled supernatantwassubjected toHPLC analysis.
Proteolytic Digestion and Purification of hC21dR Frag-ments. The immunecomplex of hC21dR and h453-TAS was
digested at37°C with trypsin(Sigma; seeTable 1). The gel waschilled onice, separated from supernatant by centrifu-gation (2000 x g, 4°C, 3min),and washed three times with
ProteolyticCharacterization of Free Antigen and the Anti-body. The structure and purity of hC21dRwas verified by
FABMS of the intact peptide and by direct analysis of the mixtureoftrypticpeptides, which yielded abundant proton-ated molecular ions [M + H]+ for peptides T1, T3, and T4 (Fig. 2). Relative cleavagerateswithtrypsinweredetermined
atconditions oflimitedproteolysis and showed rapid hydrol-ysis of peptide bonds at Arg'0 and
Arg13
and a somewhatslower cleavage at Arg8 (data not shown). However, this reaction scheme issignificantly differentuponbinding of the
antibodyasdescribed below.
Incontrast,theantibody revealedaremarkable resistance
towards proteolytic degradation. No cleavage of mAb h453
I
~~~~~~~~~~~~~~..
CNYITELR
QR HAR::
... ...ASUG
...I -T1 1 ST3j T4 1 Cu la ._ en O T4 669 -T---T3/4 Ti 1083 T3 383 i)1 825 I ..IL 1-I . I. 1 I11 400 600 800 1000 m/z
FIG. 2. Amino acid sequence and FABMS analysis oftryptic peptidesof thesynthetic antigen hC21dR.Labeledpeaksdenote[M
+H]'ions ofpeptide fragmentsT1, T3,and T4. Thesequenceshown represents[GIn65,Arg66]hC3a-(57-76).Thestippled partialsequence
representsthesynthetic octapeptideused forproductionof the mAb
h453.AAM,S-acetamidomethyl. FreeAntigen
NO
Mass SpectrometricPeptideMapping Antigen Non-Epitope EpitopePeptide
4
%
hPeptides
m/z m/ m/z
Biochemistry:
Suckauetal.Proc. Natl.Acad. Sci. USA 87 (1990) Table 1. Quantification of tryptic peptides isolated from h453-TAS-bound hC21dR at different
proteolysis conditions
mAb-bound Reaction %tryptic peptide Ratio
Trypsin,
hC21dR,
:S (wt/wt) time,rycpepe
oflug ,ug hC21dR* h453-TAS min Tlt T4+T3/T4§ T4 to
T3/T49
0.05 1.67 1:33 1:4400 20 80 ND -0.05 1.67 1:3.3 1:440 20 90 68 1:5 5 1.67 3:1 1:44 20 90 68 2:1 20 3.25 6:1 1:18 10 97 65 13:1 50 3.25 15:1 1:7 120 -11 -11 ND, notdetermined.
*E:S
formAb-bound antigen.tExpressed
asthe molar percentage of mAb-bound-hC21dR as determined by HPLC.tPeptide
T1 from supernatant.§Sum
ofpeptidesT4andT3/T4fromMgCI2dissociation.1Estimatedbyratio of [M + H]+ionabundances in PDMS. "Notdetermined due to extensivedigestion of mAb. was detectable by
SDS/PAGE
at conditions that provided complete proteolysis of the antigen [enzyme-to-substrate ratio (E:S) = 1:100 (wt/wt)]. The native mAb was notdegraded,even athighproteaseconcentrations (E:S = 1:2),
whereas heatdenaturation
led
torapid proteolytic digestion.A comparable stability of the mAb was found towards a-chymotrypsin and Staphylococcus aureus V8 protease, as
previously reported for different mAbs (6,
7),
suggesting asimilar
utility
of theseenzymesforpeptide
mappinganalysis ofimmune complexes.Molecular
weight
determination of intactgpC3a
anddes-Arg78-gpC3a
by PDMS and mass spectrometric analysis of trypticpeptide mixturesprovided
structuralinformation
con-cerning
the entiregpC3a
sequence (26).Particularly,
[M +H]+
ions of the N-terminaltryptic peptides
and peptidescontaining
residues 66-70 or residues 71-77 from the C terminus ofdes-Arg78-gpC3a
were identified inhigh
abun-dances,indicating
completecleavage
atArg65
and Arg70, which is incontrast totheepitopemapping discussed
below (seeFig. 5). Athigh
E:S(1:20),
additional molecular ions of large polypeptide fragments could beassigned
to partialdigestion
in the central part of the C3a sequence(data
notshown).
Formation and Dissociation of Immune Complexes. The
binding capacity
andspecificity
ofh453-TAS,
the dissocia-tionprocedure,
andrecoveryof intactantigen
wereevaluated with the aid of HPLC andmassspectralanalysis.In atypicalexperiment,
from 4 ,gofhC21dR bound toh453-TAScon-taining 75,gofmAb, 1.4 ,ugofantigenwasrecovered with TS
buffer,
andnofreehC21dR
wasdetectableby
HPLC in thesupernatantafter additionalTStreatment.Dissociation of theimmunecomplex
withMgCI2
ledtothe release of -1jig
of
antigen
(i.e.,
40% of the theoreticalbinding
capacity
of themAb).
After HPLCpurification,
theantigen
wasidentified
asintact hC21dR
by
the [M +H]+
ion(m/z
=2382)
and the doubly charged ion intheplasmadesorption
massspectrum(see
Fig.
4c). In acontrolexperiment
withSepharose
con-taining
tresyl groups blockedby
treatment with 0.1 MTris-HCl/0.5
MNaCI,2.4,ug
ofhC21dRwasrecovered in theTS
supernatant, andnopeptidewasdetectable in theMgCl2
eluate. Inaddition,
thespecificity
of h453-TAS was tested with a mixture of partial tryptic peptides of hC21dR and exclusively yielded peptides in theMgCl2
eluate that con-tainedtheantigenic C terminus(data
notshown).
Thebinding
and dissociation procedure could beperformed repeatedly
with the same batch of h453-TAS without loss ofbinding
capacity,
indicating
thefeasibility
of
thelyotropic
agentMgCI2
(27) forefficient
dissociation of the immobilized immunecomplex withoutaffecting
themAb's function.IdentificationofTrypticEpitope Peptidesfrom mAb-Bound
hC21dR.
Samples
of thepurified
immunecomplex
ofhC21dR
and h453-TAS were subjected to trypsin digestion using a wide range ofenzyme-to-hC21dR ratios and reaction times (Table 1). Under variousdigestion conditions, HPLC anal-yses ofsupernatants andMgC12eluatesconsistentlyyielded
major peptidefractions with retention times of 26.2 and 25.1
min,
respectively (Fig. 3). In addition, nonpeptide contami-nantcomponents were presentin thechromatogramsat21, 23.3, and 24.1 min. PDMS analysis of thepurified peptidescontained in the supernatant (i.e., nonepitope fraction)
yielded a predominant [M + H]+ ion (m/z = 1083) of fragment T1 due tocleavage atArg8(Fig. 4b). By contrast,
thespectrum of thefraction afterMgC12dissociation (Fig.4a) showed a most abundant [M + H]+ ion of the C-terminal
peptide T4 (m/z, = 669), together with a small ion ofthe
coeluting peptide T3/T4 (m/z, = 1033), which was unre-solved by HPLC. An=90% release of T1from mAb-bound
hC21dR in thesupernatantand65% release ofT4andT3/T4 from the truncatedimmune complexwerefound with E:Sas
-I I 8 q .0 0 DA .0 CU 6) 30 4.) 0 20 10 20 22 24 mm
FIG. 3. HPLC analysisof supernatant (tracesd-f) and
MgCI2-dissociatedpeptidefragments(tracesa-c)aftertrypsindigestionof h453-TAS-boundhC21dR, atdifferent protease concentrations and reaction times(see Table1). HPLC conditionswere asdescribedin Materials and Methods. Trace a, 20 ,ug oftrypsinfor10min;traces
b andd,5,ugoftrypsinfor 20min;tracescand e, 0.5jgoftrypsin
for 20min;tracef,0.05,ug oftrypsinfor 20 min. Unlabeledpeaksin thechromatogramsareduetononpeptidecontamination.
8 I a T4 669
.1]
a T3/4 1033 r-i b I I1 1083 1 (M+H) c 2382M2+
I,I.,
.J
600 1600 2600 m/zFIG. 4. PDMS analysis ofHPLC-purified hC21dR andtryptic peptides from the immune complex with h453-TAS. (a) Epitope fraction at 25.1 min in trace aofFig. 3. (b) Nonepitope peptide
fraction at 26.2min in trace dofFig. 3c. (c) IntacthC21dR after MgCI2 dissociation from the immune complex. M2' denotes the doubly protonatedmolecular ion(m/z=1191);the ionatm/z=794
is duetoanonpeptide contaminant.
high as6:1 (see Table 1). At veryhigh protease concentra-tions, specific peptide fragments were no longer detectable
because ofprogressive destruction of the mAb. Low E:S led to theformation ofincreasing amountsofT3/T4 relative to T4asestimatedby their [M+H]+ ion abundances, indicating
areduced tryptic cleavage rateatArg'3 relative to Arg8 due to steric hindranceby the mAb boundtoits epitope.
How-ever, atanyproteolytic conditions amenabletoPDMS
pep-tide mapping analysis and irrespective of nonpeptide
con-taminations, the epitope peptides T4 and T3/T4weretheonly
peptide fragments in the MgCI2 eluate. In contrast, some
contamination of thenonepitope fraction by epitope peptides
was observed, due to release of fragments from hC2ldR nonspecifically bound to the polypropylene cup surface.
Only the epitope fractionwas,therefore, used for the epitope identification ofgpC3a.
Epitope Identification from gpC3a. Proteolytic procedures and PDMS analysis were applied in the same manner to epitope mapping of des-Arg-gpC3a bound toh453-TAS. On the basis of the results obtained with theimmunecomplex of hC21dR with h453-TAS, digestion was performed at two differenttrypsin concentrations that didnotdegrade the mAb while fragmenting C3a (enzyme-to-C3a ratios, 6.5:1 and 1:1.5). In all experiments, HPLC analysis yielded a single majorpeptide fractionat23.8min uponMgCI2 dissociation.
PDMSanalysis (Fig. 5) showedapredominant [M +H]+ion (m/z = 901) of the C-terminal fragment containing residues
8
a
4) 0_
1000 2000 m/z
FIG. 5. PDMS analysis oftryptic epitope peptidesdissociated fromthe truncated immune complexofdes-Arg78-gpC3a with the
mAb h453 (ES = 6.5:1). Numbers in parentheses denote partial
sequencesofgpC3a;the ionatm/z= 1057(70-78) originatesfrom
traces ofgpC3a present. The C-terminal sequence of des-Arg78-gpC3ais shown. The C-terminal tryptic cleavage sites in the free
antigen (Arg70,openarrowhead)and in the immunecomplex (solid arrowhead)areindicated.
70-77, associated with minor ions of the peptides containing residues 66-77 and residues 70-78 ofgpC3a, due to incom-plete cleavage at Arg69 and a small amount of
non-desarginatedgpC3a,respectively. These peptides clearlycan
be named epitope peptides since they all contain the se-quencemotifLeu-Gly-Leu-Ala, which ispartoftheepitope
sequence(see Fig.2and thesequenceinFig. 5).In contrast to the cleavage at Arg70 found by tryptic digestion of free gpC3a (26), cleavage ofthe mAb-bound antigen occurred exclusivelyatArg69. Asnotedabove, this effect isin
accord-ancewiththe stericrequirements of trypsin binding (28)and withinaccessibility of Arg70 duetobinding of the mAbtoits epitope approximately three amino acid residues
down-stream.
Moreover, ELISA data yielded a30-fold loweraffinityof
h453 for des-Arg78-gpC3a as compared to des-Arg77-hC3a (data not shown), which strongly indicatessequencevariation within the epitope region. In addition to thesequence motif
Leu-Gly-Leu-Ala, which is conserved in both species andis responsible for the immunological cross-reactivity of h453,the epitopeondes-Arg77-hC3amusttherefore containone orboth of theevolutionary variant residues, His72 and Ser71. On the other hand, the weak exopeptidase activity of trypsin and crystal structure data of trypsin inhibitor complexes (28) indicatebinding approximatelytworesidues downstreamasa
prerequisite to tryptic cleavage at Arg69 in the gpC3a se-quence. Supported by these additional data, the mass
spec-trometric results provide evidence forHis-Leu-Gly-Leu-Ala [i.e., hC3a-(72-76)] being the epitope recognized by h453,with Ser71as apossiblepartof theepitopesequence.
CONCLUSIONS
The approach described in this study presents several ad-vantages to the molecular characterization ofepitopes by combining the selectivity of partial proteolysis of immune complexes with the molecular specificity of accurate mass
65 70 75 1
-L-R-Q-Q-H-R-R-E-Q-N-L-G-L-A|
(70-in 901(70
-78)
(66-77)
1057 1450 m lbr
Proc. Natl. Acad. Sci. USA 87 (1990) spectrometric molecular weight determination. Irreversible
immobilization of the mAb and analysis of the bound peptides after proteolytic digestion provide the basis to name the selectively dissociated fragments epitope peptides.
Important prerequisites for accurately defining epitope structures are(i) high affinity of the proteolytic peptides (i.e., truncated antigen) to the mAb and (ii) the availability and
accessibility of suitable proteolytic cleavage sites. Despite somesuppression by the mAb, tryptic cleavage was shown to occurapproximately two amino acid residues away from the epitope. At least two other endoproteases, a-chymotrypsin and S. aureus V8 protease, appeartobe well suitedand could besupplemented by exopeptidases to further define epitope sequences. Epitope extraction and direct identification of peptide fragment mixtures by PDMS was shown to be applicable to the analysis of a sequential epitope and may
enable the characterization ofevenconformationally defined
and assembled topographic epitopes, which are not detect-ableby synthetic peptides as antigenic probes. In the course of an exactdescription of an epitope (e.g., using site-directed mutagenesis), its application will greatly reduce the number ofpossible antigenic residues to be tested. Thus, epitope
extraction and mass spectrometric peptide mapping of a single immune complex represents a sensitive and rapid
methodofhighmolecularspecificityin theanalysis of protein antigens.
Wethank R. Burgerfor the generous gift of the mAb h453 and R. Gerardy-Schahn for stimulating discussions. This work was sup-ported by grants from the DeutscheForschungsgemeinschaft,Bonn, F.R.G. (Pr 175/2), by the Bundesministerium fur Forschung und Technologie, Bonn (01VM89014), and by the University of Kon-stanz.
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