- Email: [email protected]
Characterization of protein-hapten conjugates by mass spectrometry
C. R. Acad. Sci. Paris, t. 1, S&ie II c, p. 35-40, Chimie bioorganique et thCrapeutique/Bioorganic
1998 and medicinal
chemistry
Characterization of protein-hapten conjugates by mass spectrometry Nelly GOURLAOU~N”, Florence GONNETb,
GCrard BOLBACHb*, And&e MARQUET”
‘I I.aboratoire de chimie
organique Paris cedex 05, France
75252
lbiologique.
URA
CNRS
.3 November
1997,
accepted
I December
FLORENTIN”,
403, universid
Paris-VI,
4, placeJussieu,
f-1’ CIGRS
103, universitk
Paris-W,
” Laboratoire de chimie structurale organique et biologique. 4, place Jussieu, 75252 Paris cedex 05, France F.-mail : [email protected]. (Received
Dominique
tour 44-45, b%timent
boEte 182.
F, boite
45,
1997)
Abstract
- An hapten 1 designed for the production of catalytic antibodies was synthesized after coupling its precursor 2 to bovine serum albumin (BSA). The conjugates BSA-2 and BSA-1 were characterized by MALDI This paper shows that besides the average number of molecules bound to the proteilr mass spectrometry. deduced from the molecular ion peak shift, the MALDI technique can also give access to their distribution, b! sirnulation of the peak. protein-hapten
RPsumC conqu &rum
pour
conjugates
/ distribution
- CaractCrisation la production
albumine
/ mass
de conjuguCs d’anticorps
/ MALDI-TOF
spectrometry
protkine-haptike
catalytiques
par spectromktrie
a &tt! synthCtis6
de bceuf (BSA). Cette note montre que la technique
au nombre merit, par
moyen de moltcules li6es B la proteine simulation du pit B leur distribution.
conjug&
protkine-hap&e
Version franqaise
! distribution
dPduit
/ spectromktrie
apr&s
couplage
MALDI
du dPplacement
de masse
de masse. L’hapGne de son
I, 2 sur la
prCcurseur
peut donner accts, non seulement
du pit
de I’ion
mol&culaire,
mais
&gale-
/ MALDI-TOF
ab&gCe
Les petites moltcules exigent, pour ttre immunog&es, un couplage covalent avec une prottine. LJ caractkrisation des conjuguks protkines-hapdnes ainsi obtenus reste toujours un probltme diffkilc comme I’attestent un certain nombre de revues rkcentes [ 1, 21. La spectromCtrie de masse (SM), avec la mise au point ces dernikres an&es de nouvelles mkthodes d’ionisation, 1’Clectronkbulisation (ESI) et la dksorption-ionisation laser assist&e par matrice (MALDI), apparait comme un outil trts prometteur pour une telle caractkrisation [ 1, 21. A priori, le couplage HPLC-ESI-SM semble le plus appropriC pour dtterminer la distribution d’haptenes fix& B la protkine [l, 21. Nous montrons dans cette note que ie mode d’ionisacion MALDI, combine B la SpectromCtrie masse temps-de-vol, peut kgalement permettre, par une analyse prkcise des don&es et l’utilisation techniques de simulation, de determiner cette distribution.
Communicated - correspoldme
125 I-8069l9tt8/00010035
by Ftanpis
de de
MATHEY.
and rcprir,ts.
0 Ad
Cmie des Sciences/EIsrvier,
Paris
35
N. Courlaouiin
et al.
Les conjuguis que nous analysons ici ont Ctk conGus pour tester des anticorps catalytiques. La rCaction que nous avons choisi de mimer est la carboxylation du ribulose 1,5-biphosphate, rCaliste dans les plantes (sch&zti 1) par la ribulose-l,5-biphosphate carboxylase oxygtnase (Rubisco) [4, 51. La synth&se de l’haptene 1, analogue d’un inhibiteur connu de la Rubisco, le carboxy arabinitol-I ,5-biphosphate [6], est d&rite par ailleurs [3]. BSA-1 est obtenu par carboxylation de BSA-2, ce dernier provenant du couplage de la BSA avec 2 (schhma 1). BSA-1 et BSA-2 n’ont pu &re caract&isCs en ESI. La perte de r&olution et le trts faible rapport signal/bruit constituent toutefois une preuve d’une distribution t&s large d’haptenes fixes sur la BSA. L’analyse en spectrom&rie de masse MALL11 de BSA, BSA-1 et BSA-2 montre la prtsence en mode ion positif des ions molCculaires. Les pits des ions moltculaires de BSA-2 et BSA-1 sont deplac& respectivement de 3 318 + 30 Da et de 3 530 * 30 Da par rapport B celui de la BSA (Jlgure I). De plus, 2 505 + 95 Da et 2 754 f 19 Da sont beaucoup les plus Cleleur largeur B mi-hauteur, respectivement vies que celle de la BSA (1 133 k 18 kPa). Dans l’hypothese oti les facteurs qui dgfinissent le centroi’de du pit [7, 81 de BSA restent inchan@ lorsque 1 et 2 sont attach& B la BSA, le decalage en masse observC permet de deduire le nombre moyen d’haptknes fix& par molCcule de BSA : 10,5 pour BSA-2 et 3,8 pour BSA-1. Dans cette hypoth&e, il est possible de remonter 4 la distribution d’hapdnes, en remarquant que si n haptenes sont fix& ?I une moltcule de BSA, le pit correspondant sera celui de la molCcule BSA, observt exptrimentalement, mais d&al& en masse de n fois la masse de la molPcule d’haptkne fix&e (m = 3 12,2 Da pour BSA-2 et m = 358,2 Da pour BSA-l), 1es modifications de composition isotopique restant ntgligeables. Pour une distribution a(n) d’haptenes, I’intensitC de ce pit seraproportionnelle B a(n). Le pit mol&ulnire de BSA peut &re decrit de faGon satisfaisante par une distribution gaussienne (&UW 2a). Cette distribution sert de point de dkpart pour la simulation des pits des ions molPculaires de BSA-1 et BSA -2. La distribution d’haptenes a(n) est alors ajust@e de faGon g ce que les pits simul& et les pits exp&irnentaux se recouvrent au mieux. Les$$res 26 et 2r montrent respectivement pour BSA-2 la comparaison du pit calculi? (meilleur ajustement) et du pit experimental, et le d&ail de la distribution d’hapti:nes correspondante. La distribution d’haptbne est gaussien ne et est cent&e sur IZ,~= 10,i mol&cules d’hapttnes par molCcule de BSA (variance G”‘= 8,2). Lajggre 3a montre le r6ultat de la simulation du pit de I’ion m&culaire de BSA-1, en considPrant la m&me dlistribution d(n) que celle prtctdemment determince pour BSA-2. On peut remarcluer que l’ajustement avec le pit expdrimental est loin d’&tre satisfaisant, puisque le pit de simulation est d&place vers les hautes masses. Ce rtsultat indique, soit une transformation incomplkte de BSA-2 en BSA-1, soit une lactonisation de l’haptene dans la solution de matrice (srhhm~ I). Le meilleur ajustement pour BSA-1 sous forme non modifiee et sous forme lactone correspond encore B une distribution gauss&me d’haptknes cent&e respectivemenr sur no = 10,O (oo’ = 8,7) et n,, = lo,2 (Oar’ = 8,7). On peut noter que ces valeurs restent t&s pro&es. Elles sont en bon accord avec les estimations faites en utilisant un traceur radioactif [‘“CIKCN dans la formation de
BSA-1 [3].
1. Introduction Small molecules require covalent bonding to a protein to become immunogenic. The characterization of such protein-hapten conjugates is still a challenging problem, as pointed out in recent interesting papers reviewing this topic [l, 21. Besides more classical techniques (UV, radiolabelling, etc.), mass spectrometry appears as a very promising tool, as shown by Adamczyk et al. [ 1, 21. However, there are only a few reports on the use of this technique and we present in this work a further example. Mass
36
spectrometry can give access to the distribution of haptens linked to the protein using LC-Electrospray-MS [2] but only to the mean number of haptens in MALDI-MS [I]. However, for large hapten distributions, the classical Electrospray mass spectra are complex since they involve multiple protein species in vario’us multicharged states and deconvolution of the mabs spectra is difficult. According to Adamczyk et al. [2], only an in-line coupling LC-Elcctrospray Mass Spectrometry allows to get an interpretable deconvoluted mass spectrum giving information on the hapten di.\tribution.
Mass
In this work, we will show that the study of all the MALDI mass spectra data (mass shift and broadening of the molecular ion peak) allows in conjunction with simulation techniques to deduce the hapten distribution. A good agreement was observed on the hapten distribution studied by MALDI-MS and the mean number obtained by radiolabelling techniques [3]. The conjugates that we are analyzing here were designed to screen catalytic antibodies. The design and the synthesis of the hapten will be described in details elsewhere [3]. Compound 1 [4, 51, chosen as hapten, is an analogue of the well-known excellent inhibitor of Rubisco: carboxyarabinitol-1,5-bisphosphate [6] (scheme I). Its precursor 2 was first coupled to BSA and the transformation of the keto group into the hydroxy acid moiety was achieved on BSA-2 to give BSA-1.
spectrometry
of protein-hapten
conjugates
1 solutions were typically at concentrations ot 2 x 1Om5to 10m4 M. The matrix solution was mixed respectively with BSA, BSA-2 and BSA1 solution in order to get a protein concentration of - 10 pmol/uL ( matrix-to-analyte molar ratio: lo4 to 5 X 104). Finally, 0.5 to 1 pL of the mixture was deposited onto a clean gold sample holder and allowed to dry in air at room temperature. Positive ion mass spectra went obtained using a laser irradiation near the threshold of the BSA molecular ion emission. Each mass spectrum corresponded to an average of 256 laser shots. The data concerning the centroid and the full width at half maximum (FWHM) of the proteins were obtained frsom the analysis of 10-l 5 mass spectra recorded OII various samples. The samples prepared as men-tioned above were homogeneous.
2.1. Formation of the BSA-2 conjugate 2. Experimental
section
MALDI mass spectra were obtained on a Time of Flight Mass spectrometer Voyager Elite (PerSeptive Biosystems, Framingham, MA) equipped with a pulsed N; laser and an ion delay extraction. Due to the high masses of the studied proteins, the instrument was tuned in the linear mode. Sinapinic acid was used as matrix at a concentration of 10-l M in acetonitrile/water 411 (v:v). BSA, BSA-2 and BSA-
$H20P0: HO-
&COO
CH20H -
‘/ F-OH
H-&OH
BSA was purchased from Sigma (Fraction V, powder A2934 lot (93AO231)). A solution of2 (15 mg, 45 pmol) in 0.83 mL of water wa\ added to a solution of BSA (50 mg, 0.75 pmol) in water (2.4 mL). A solution of EDC (43 mg. 22,O umol) in 1 mL of water was added. l’hc mixture was stirred overnight at 4 “C. The solution was divided into three fractions of 1.26 ml, each and one of 0.41 mL, which were dialyseti 12. h in 2 1. of water.
vH20H COO-
y=o HO-F-H
HO-q-H HO-F-H
H-&-OH kH20PO:‘
HO-F-H
CH,0P03(CH2),C00 (D) Carboxyarabinitol 1,5-bisphosphate
1
I;H20H
yy?oo -
c HO-
+-OH C-H
HO-
6-H
~H20P03(cH,),c0-~~~~~
kH20P03(cti2),c0-~~~~~ BSA-1
Scheme
1. Synthesis
of hapten-BSA
SchCma
1. Synthkse
des conjuguk
2
i e
BSA EDC
TH>OH
KCN HO-
q=o q-H
HO-$-H CHeOP03(CHp)&O-NHBSA BSA-2
conjugates. haptkes+BSA.
37
et
N. Gourlaou&n
2.2. Formation
al.
of the BSA-I
conjugate
A 0.15 M aqueous solution of KCN (45 uL, 6.9 umol) was added to a solution of BSA-2 conjugate (15 mg, 0.23 pmol) in I mL ofwater. The mixture was stirred 48 h at 4 “C and dialysed in 1 L of water overnight.
3. Results
and discussion
The BSA and BSA-1 conjugates were analyzed by ES1 mass spectrometry. The resolution was completely lost for the conjugates providing evidence of a large distribution of attached molecules. It was not possible, due to the very low signal-to-noise ratio, to extract any reliable information on the hapten distribution. BSA, BSA-2 and BSA-1 were characterized by MALDI mass spectrometry. Figure 1 shows typical partial positive ion massspectra of BSA, BSA-2 and BSA-1 using an external calibration. The molecular ion peaks of BSA-2 and BSA-1 were shifted toward the high-mass range by 3 3 I8 f 30 Da and 3 530 + 30 Da respectively. Their full width at half maximum (FWHM) strongly increasedwith respect to the
FWHM of the molecular ion peak of BSA, 1133* 18Da,2505+95Daand2754rt 13 Da respectively. A shift and a broadening were also observed on the doubly charged BSA-2 and BSA-1 ion peaks. From the massshift, a mean number of molecules of 2 and 1 attachecl to BSA can be deduced, similarly to what was described by Adamczyk et al. [l, 21. This mean number is respectively 10.5 and 9.8 for BSA-2 and BSA-1. The assumption, from which this mean number can be deduced in a relevant manner implies that the factors influencing the mass centroid [7,8] of BSA are not modified for BSA with attached 1 or 2. According to this assumption, an homogeneous attachment of r/ molecules to BSA leadsto a peak shifted to the highmass range by n x mass of the molecule, the peak width being unchanged. Thus, for a given distribution of 1 or 2 attached to BSA, it ispossible, using the BSA experimental peak, to sim-ulate the peak expected for BSA-2 and BSA-1 and to compare to the experimental data. For practical reasons, the BSA experimental peak was simulated by a gaussian distribution, in order to calculate, for a given mass,the contri-
BSA-1
L 60000
62000
64000
66000
68000
70000
72000
74000
76000
781
Mass(m/z)
Figure 1. MALDI partialpositivemass spectra ofBSA, BSA-2 .and BSA-1 in the BSA-1 mass spectra were obtained at higher laser energy (2.8 pJ/pulse) than that the mass corresponded to an average of5 different mass spectra on a same target. averaging of 256 lasers short (external calibration using horse skeletal muscle
region ofthe molecular ion peak. I!&\-2 and ofthe BSA mass spectrum (2.1 pJ/pulse). ~11 A mass spectrum was obtained with .s signal apomyoglobin).
Figure 1. Spectre dr masse MALDI de BSA, USA-2 ct BSA--1 montrant la region des ions molt?ulaires. Les specrrea de BSA-2 ec BSA-1 ont et& obtenus pour une Cnergie du pulse laser (2,8 pJ/pulse) plus GlevCe que celle ut.ilis& pour le specrre de BSA (2,l pJ/pulsc). Les spectres montrCs r&ultrnt d‘une moyenne de cinq spectres de masse rt?alis& sur un mCme &hantillon. Chaquc spectre de masse r&ulte du signal moyrn nbtenu j partir de I’irradiation de I’Cchantillon par 25(, tils laser.
38
Mass
bution of each conjugate to the stimulate peak. All these calculations were performed using the sofrware KaleidaGraph (version 3.0.2). As shown injgure2(a) a gaussianfit can be usedto satisfactorily describe the BSA experimental peak, except in the higher-mass range. Under the above assumption, the peak of BSA with n attached haptens should be gaussianand shifted by n X m (m = 312.2 Da for BSA-2 and m = 358.2 Da for BSA-l), the intensity of this peak being proportional to the relative abundance a(n) of,BSA with n attached haptens. The distribution a(n) was adjusted to get the best fit of the experimental peak. Such a procedure was recently applied by Tang et al. [9] to MALDI quantification of P-2-microglobulin glycosylated end products in human serum. Figure 2(b) and (c) respectively show the best simulation for the BSA-2 peak and the contribution of the different BSA-2 species.Only the most abundant species have been shown in figure 2(c). For BSA-2, the distribution of 2 extracted from this simulation is quite well described by a gaussian one centered on no = 10.5 molecules of 2/BSA molecule (mean square deviation (5()* = 8.2). Obviously, n,, is equal to the mean number of conjugates deduced from the experimental mass shift between BSA and BSA-2.
spectrometry
of protein-hapten
conjugates
Figure 3(a) shows a simulation of the BSA-1 peak using the distribution a(n) previously determined for BSA-2. The simulated peak does not fit correctly the experimental peak since it is shifted toward higher mass. This could indicate that the transformation of BSA2 to BSA-I is not complete. Another possibility would be to consider a lactonization of the hapten in the acidic matrix solution (schemeli. F’gure 3(b) and (c) show the best fits respectively for BSA-1 and the lactone form of BSA1. In both cases,the hapten distributions were gaussianand centered respectively on no = 10.0 (oo’ = 8.7) and tie =10.2 (o,,’ = 8.7). These values are closeenough, taking into account the experimental uncertainties, to conclude that: BSA-2 has been transformed almost completely into hapten, showing that this simulation approach can be very useful to follow a chemical reaction carried out on a conjugate.
The hapten quantification was also achieved with a radioactive tracer [14C]-l. After exrensive dialysis to free the conjugate from the excessKCN, the remaining radioactivity on the protein was counted. This method gives 9 * 1 molecules of l/BSA molecule [3] in good agreement with the MALDI results. This good agreement shows that the assumption used for the simulation is quite reasonableand that all 1 lo2
1on
r
60
65000
66000
67000
68000
69000
70000
64000
nla*s
66000
68000
70000 72000 nlass
74000
76000
781100
Figure 2. (a) Experimental BSA peak (-) and simulation (- - -) by a gaussian distribution. (b). Experimental BSA-2 peak (-) and the best simulation (- - -) using a gaur.sian distribution of2 (see text). (c). Details of the hapten diatribution for BSA-2. The upper curve corresponds to the sum of all the BSA-2 contributions.
640M)
66000
68000
70000 72000 ln‘lss
74000
76000
78000
Figure simule masse lation (- -) pour toutes
2. (a) Pit de masse exp&imenral de BSA (-) et pit par one distribution Sausienne (- -). (b) Pit de exp&imental de BSA-2 (-) et la meilleure simuobtenue B partir d‘une distribution gausienne de 2 (voir texts). (c) D&ails de la distribution d’hapt&cy RSA-2. I a courbe sup&ieure indique la somme de Its contributions de BSA-2.
N. Gourlaouih
et al.
80 60
68000
72000
76000
mass
68000
64000
72000 mass
76000
Figure 3. (a) Experimental
8 10’ no=10.2
olP=n.7
f!! 610’
x .2
BSA-1 peak (-) and simulation (- -) uring the hapten distriburion found for BSA2 (see rext andjgureZ(c)). (b) Experimental BSA-1 peak I -) and the besr simulation (---) (see rexr). (c) Experimental BSA-I peak (--) and rhe besr simulation (- - -) assuming a complete lacronizarion of BSA-1 (see rexlj.
Figure 3. (a) Pit de masse exp6rimencal
2z 4 10' IE 2 10’ 0 lu” 64000
68000
72000 mass
76000
the factors implied in dze mass centroid and the width of the BSA peak (matrix and/or cat-ion adducts, ion initial velocity distribution, metastable decomposirion 171) are nor srrongiy modified when hapten is attached. Further experiments on other protein conjugates including the comparison between different techniques (radioactivity, ESI-MS) would be very useful to further validate the MALI31 approach. This technique could be quite general and
applied aswell for instance ta the srudy of rhe distribution
of post-translational
modification.
simulation pour BSA-2 p6rimental obrenue (-BSA-1 (-) posanr une
de BShl (-) er (-- -) utilisanr la disrribution d’haptenes trouv& (voir texte erfigun ,? (c)). (b) Pit de masse exde BSA-1 (-) et la meilleure simularion -) (voir rexrel. (c) Pit de masse exp&imcntal de er la meilleure simularion obtenue (- -) en suplacronisacion complPre de BSA-1 (voir texrc).
[3] (a) Gourlaouen Paris, 1995. (b) Gourlaouen ted.
(b) Harrman (1394)
(b) Pierce J., Andrew (1986)
Chem
40
D., Marquer G.H.,
M.R.,
Ann.
Ann.
A.. ,ubmitRev. Bwchem.
Rev. Biochem.
T.J., Pierce 1.. Schlws B 313 (1986) 397.
T.J., Lorimer
Jaworowski A., Hartman Chcm. 259 (1984) 6783.
CC)
[6] I’iercc
J., Toolbert 934.
[81 Beavis R.C.,
63
G.H.,
J.V.,
1. Biol. Chern.
N.E.,
F.C.,
Barker
lttst
I.A.,
I. Riol.
R., Biochemistry
A., Karas M., Hillekamp
19
F.. Giessman IJ., lnt. .$45-354.
Spsctrom. Ion Processes 1.11 (1994) Chair
B.T., Anal. Chrm.
62 (1990)
1836
1840. [91 Tang X., Sadeghi
P.C., Biowniuga:arc
P-et-M.-Curie,
10248.
[I] AdamcLyk M., Buko A., Chen Y.Y.. Fishpaugh J.R., Gebler J.C., Johnson D.D., Rioconjugate Chcm. i M., Gehler J.C.. Mattingly 7 (I w6) 475-481
EC., Harpel
G.H., Andrew Phil. Trans. R. Sot. Land.
171 Ingendoh J. Mass
631-635.
Lorimcr
[51 (a) Lorimer
261
University
197.
References
(1794)
Thesis
N., Florentin
141 (a) Mizmrko H.M., 52 (1983) 507.
(1980)
[2] AdamcLyk
N.,
Mcllwain 3740-3745.
L.K.,
M., Olumec Z., Verres A., Rraatz J.A., Dreifuss P., Anal. Chem. 68 119%)
1998 and medicinal
chemistry
Characterization of protein-hapten conjugates by mass spectrometry Nelly GOURLAOU~N”, Florence GONNETb,
GCrard BOLBACHb*, And&e MARQUET”
‘I I.aboratoire de chimie
organique Paris cedex 05, France
75252
lbiologique.
URA
CNRS
.3 November
1997,
accepted
I December
FLORENTIN”,
403, universid
Paris-VI,
4, placeJussieu,
f-1’ CIGRS
103, universitk
Paris-W,
” Laboratoire de chimie structurale organique et biologique. 4, place Jussieu, 75252 Paris cedex 05, France F.-mail : [email protected]. (Received
Dominique
tour 44-45, b%timent
boEte 182.
F, boite
45,
1997)
Abstract
- An hapten 1 designed for the production of catalytic antibodies was synthesized after coupling its precursor 2 to bovine serum albumin (BSA). The conjugates BSA-2 and BSA-1 were characterized by MALDI This paper shows that besides the average number of molecules bound to the proteilr mass spectrometry. deduced from the molecular ion peak shift, the MALDI technique can also give access to their distribution, b! sirnulation of the peak. protein-hapten
RPsumC conqu &rum
pour
conjugates
/ distribution
- CaractCrisation la production
albumine
/ mass
de conjuguCs d’anticorps
/ MALDI-TOF
spectrometry
protkine-haptike
catalytiques
par spectromktrie
a &tt! synthCtis6
de bceuf (BSA). Cette note montre que la technique
au nombre merit, par
moyen de moltcules li6es B la proteine simulation du pit B leur distribution.
conjug&
protkine-hap&e
Version franqaise
! distribution
dPduit
/ spectromktrie
apr&s
couplage
MALDI
du dPplacement
de masse
de masse. L’hapGne de son
I, 2 sur la
prCcurseur
peut donner accts, non seulement
du pit
de I’ion
mol&culaire,
mais
&gale-
/ MALDI-TOF
ab&gCe
Les petites moltcules exigent, pour ttre immunog&es, un couplage covalent avec une prottine. LJ caractkrisation des conjuguks protkines-hapdnes ainsi obtenus reste toujours un probltme diffkilc comme I’attestent un certain nombre de revues rkcentes [ 1, 21. La spectromCtrie de masse (SM), avec la mise au point ces dernikres an&es de nouvelles mkthodes d’ionisation, 1’Clectronkbulisation (ESI) et la dksorption-ionisation laser assist&e par matrice (MALDI), apparait comme un outil trts prometteur pour une telle caractkrisation [ 1, 21. A priori, le couplage HPLC-ESI-SM semble le plus appropriC pour dtterminer la distribution d’haptenes fix& B la protkine [l, 21. Nous montrons dans cette note que ie mode d’ionisacion MALDI, combine B la SpectromCtrie masse temps-de-vol, peut kgalement permettre, par une analyse prkcise des don&es et l’utilisation techniques de simulation, de determiner cette distribution.
Communicated - correspoldme
125 I-8069l9tt8/00010035
by Ftanpis
de de
MATHEY.
and rcprir,ts.
0 Ad
Cmie des Sciences/EIsrvier,
Paris
35
N. Courlaouiin
et al.
Les conjuguis que nous analysons ici ont Ctk conGus pour tester des anticorps catalytiques. La rCaction que nous avons choisi de mimer est la carboxylation du ribulose 1,5-biphosphate, rCaliste dans les plantes (sch&zti 1) par la ribulose-l,5-biphosphate carboxylase oxygtnase (Rubisco) [4, 51. La synth&se de l’haptene 1, analogue d’un inhibiteur connu de la Rubisco, le carboxy arabinitol-I ,5-biphosphate [6], est d&rite par ailleurs [3]. BSA-1 est obtenu par carboxylation de BSA-2, ce dernier provenant du couplage de la BSA avec 2 (schhma 1). BSA-1 et BSA-2 n’ont pu &re caract&isCs en ESI. La perte de r&olution et le trts faible rapport signal/bruit constituent toutefois une preuve d’une distribution t&s large d’haptenes fixes sur la BSA. L’analyse en spectrom&rie de masse MALL11 de BSA, BSA-1 et BSA-2 montre la prtsence en mode ion positif des ions molCculaires. Les pits des ions moltculaires de BSA-2 et BSA-1 sont deplac& respectivement de 3 318 + 30 Da et de 3 530 * 30 Da par rapport B celui de la BSA (Jlgure I). De plus, 2 505 + 95 Da et 2 754 f 19 Da sont beaucoup les plus Cleleur largeur B mi-hauteur, respectivement vies que celle de la BSA (1 133 k 18 kPa). Dans l’hypothese oti les facteurs qui dgfinissent le centroi’de du pit [7, 81 de BSA restent inchan@ lorsque 1 et 2 sont attach& B la BSA, le decalage en masse observC permet de deduire le nombre moyen d’haptknes fix& par molCcule de BSA : 10,5 pour BSA-2 et 3,8 pour BSA-1. Dans cette hypoth&e, il est possible de remonter 4 la distribution d’hapdnes, en remarquant que si n haptenes sont fix& ?I une moltcule de BSA, le pit correspondant sera celui de la molCcule BSA, observt exptrimentalement, mais d&al& en masse de n fois la masse de la molPcule d’haptkne fix&e (m = 3 12,2 Da pour BSA-2 et m = 358,2 Da pour BSA-l), 1es modifications de composition isotopique restant ntgligeables. Pour une distribution a(n) d’haptenes, I’intensitC de ce pit seraproportionnelle B a(n). Le pit mol&ulnire de BSA peut &re decrit de faGon satisfaisante par une distribution gaussienne (&UW 2a). Cette distribution sert de point de dkpart pour la simulation des pits des ions molPculaires de BSA-1 et BSA -2. La distribution d’haptenes a(n) est alors ajust@e de faGon g ce que les pits simul& et les pits exp&irnentaux se recouvrent au mieux. Les$$res 26 et 2r montrent respectivement pour BSA-2 la comparaison du pit calculi? (meilleur ajustement) et du pit experimental, et le d&ail de la distribution d’hapti:nes correspondante. La distribution d’haptbne est gaussien ne et est cent&e sur IZ,~= 10,i mol&cules d’hapttnes par molCcule de BSA (variance G”‘= 8,2). Lajggre 3a montre le r6ultat de la simulation du pit de I’ion m&culaire de BSA-1, en considPrant la m&me dlistribution d(n) que celle prtctdemment determince pour BSA-2. On peut remarcluer que l’ajustement avec le pit expdrimental est loin d’&tre satisfaisant, puisque le pit de simulation est d&place vers les hautes masses. Ce rtsultat indique, soit une transformation incomplkte de BSA-2 en BSA-1, soit une lactonisation de l’haptene dans la solution de matrice (srhhm~ I). Le meilleur ajustement pour BSA-1 sous forme non modifiee et sous forme lactone correspond encore B une distribution gauss&me d’haptknes cent&e respectivemenr sur no = 10,O (oo’ = 8,7) et n,, = lo,2 (Oar’ = 8,7). On peut noter que ces valeurs restent t&s pro&es. Elles sont en bon accord avec les estimations faites en utilisant un traceur radioactif [‘“CIKCN dans la formation de
BSA-1 [3].
1. Introduction Small molecules require covalent bonding to a protein to become immunogenic. The characterization of such protein-hapten conjugates is still a challenging problem, as pointed out in recent interesting papers reviewing this topic [l, 21. Besides more classical techniques (UV, radiolabelling, etc.), mass spectrometry appears as a very promising tool, as shown by Adamczyk et al. [ 1, 21. However, there are only a few reports on the use of this technique and we present in this work a further example. Mass
36
spectrometry can give access to the distribution of haptens linked to the protein using LC-Electrospray-MS [2] but only to the mean number of haptens in MALDI-MS [I]. However, for large hapten distributions, the classical Electrospray mass spectra are complex since they involve multiple protein species in vario’us multicharged states and deconvolution of the mabs spectra is difficult. According to Adamczyk et al. [2], only an in-line coupling LC-Elcctrospray Mass Spectrometry allows to get an interpretable deconvoluted mass spectrum giving information on the hapten di.\tribution.
Mass
In this work, we will show that the study of all the MALDI mass spectra data (mass shift and broadening of the molecular ion peak) allows in conjunction with simulation techniques to deduce the hapten distribution. A good agreement was observed on the hapten distribution studied by MALDI-MS and the mean number obtained by radiolabelling techniques [3]. The conjugates that we are analyzing here were designed to screen catalytic antibodies. The design and the synthesis of the hapten will be described in details elsewhere [3]. Compound 1 [4, 51, chosen as hapten, is an analogue of the well-known excellent inhibitor of Rubisco: carboxyarabinitol-1,5-bisphosphate [6] (scheme I). Its precursor 2 was first coupled to BSA and the transformation of the keto group into the hydroxy acid moiety was achieved on BSA-2 to give BSA-1.
spectrometry
of protein-hapten
conjugates
1 solutions were typically at concentrations ot 2 x 1Om5to 10m4 M. The matrix solution was mixed respectively with BSA, BSA-2 and BSA1 solution in order to get a protein concentration of - 10 pmol/uL ( matrix-to-analyte molar ratio: lo4 to 5 X 104). Finally, 0.5 to 1 pL of the mixture was deposited onto a clean gold sample holder and allowed to dry in air at room temperature. Positive ion mass spectra went obtained using a laser irradiation near the threshold of the BSA molecular ion emission. Each mass spectrum corresponded to an average of 256 laser shots. The data concerning the centroid and the full width at half maximum (FWHM) of the proteins were obtained frsom the analysis of 10-l 5 mass spectra recorded OII various samples. The samples prepared as men-tioned above were homogeneous.
2.1. Formation of the BSA-2 conjugate 2. Experimental
section
MALDI mass spectra were obtained on a Time of Flight Mass spectrometer Voyager Elite (PerSeptive Biosystems, Framingham, MA) equipped with a pulsed N; laser and an ion delay extraction. Due to the high masses of the studied proteins, the instrument was tuned in the linear mode. Sinapinic acid was used as matrix at a concentration of 10-l M in acetonitrile/water 411 (v:v). BSA, BSA-2 and BSA-
$H20P0: HO-
&COO
CH20H -
‘/ F-OH
H-&OH
BSA was purchased from Sigma (Fraction V, powder A2934 lot (93AO231)). A solution of2 (15 mg, 45 pmol) in 0.83 mL of water wa\ added to a solution of BSA (50 mg, 0.75 pmol) in water (2.4 mL). A solution of EDC (43 mg. 22,O umol) in 1 mL of water was added. l’hc mixture was stirred overnight at 4 “C. The solution was divided into three fractions of 1.26 ml, each and one of 0.41 mL, which were dialyseti 12. h in 2 1. of water.
vH20H COO-
y=o HO-F-H
HO-q-H HO-F-H
H-&-OH kH20PO:‘
HO-F-H
CH,0P03(CH2),C00 (D) Carboxyarabinitol 1,5-bisphosphate
1
I;H20H
yy?oo -
c HO-
+-OH C-H
HO-
6-H
~H20P03(cH,),c0-~~~~~
kH20P03(cti2),c0-~~~~~ BSA-1
Scheme
1. Synthesis
of hapten-BSA
SchCma
1. Synthkse
des conjuguk
2
i e
BSA EDC
TH>OH
KCN HO-
q=o q-H
HO-$-H CHeOP03(CHp)&O-NHBSA BSA-2
conjugates. haptkes+BSA.
37
et
N. Gourlaou&n
2.2. Formation
al.
of the BSA-I
conjugate
A 0.15 M aqueous solution of KCN (45 uL, 6.9 umol) was added to a solution of BSA-2 conjugate (15 mg, 0.23 pmol) in I mL ofwater. The mixture was stirred 48 h at 4 “C and dialysed in 1 L of water overnight.
3. Results
and discussion
The BSA and BSA-1 conjugates were analyzed by ES1 mass spectrometry. The resolution was completely lost for the conjugates providing evidence of a large distribution of attached molecules. It was not possible, due to the very low signal-to-noise ratio, to extract any reliable information on the hapten distribution. BSA, BSA-2 and BSA-1 were characterized by MALDI mass spectrometry. Figure 1 shows typical partial positive ion massspectra of BSA, BSA-2 and BSA-1 using an external calibration. The molecular ion peaks of BSA-2 and BSA-1 were shifted toward the high-mass range by 3 3 I8 f 30 Da and 3 530 + 30 Da respectively. Their full width at half maximum (FWHM) strongly increasedwith respect to the
FWHM of the molecular ion peak of BSA, 1133* 18Da,2505+95Daand2754rt 13 Da respectively. A shift and a broadening were also observed on the doubly charged BSA-2 and BSA-1 ion peaks. From the massshift, a mean number of molecules of 2 and 1 attachecl to BSA can be deduced, similarly to what was described by Adamczyk et al. [l, 21. This mean number is respectively 10.5 and 9.8 for BSA-2 and BSA-1. The assumption, from which this mean number can be deduced in a relevant manner implies that the factors influencing the mass centroid [7,8] of BSA are not modified for BSA with attached 1 or 2. According to this assumption, an homogeneous attachment of r/ molecules to BSA leadsto a peak shifted to the highmass range by n x mass of the molecule, the peak width being unchanged. Thus, for a given distribution of 1 or 2 attached to BSA, it ispossible, using the BSA experimental peak, to sim-ulate the peak expected for BSA-2 and BSA-1 and to compare to the experimental data. For practical reasons, the BSA experimental peak was simulated by a gaussian distribution, in order to calculate, for a given mass,the contri-
BSA-1
L 60000
62000
64000
66000
68000
70000
72000
74000
76000
781
Mass(m/z)
Figure 1. MALDI partialpositivemass spectra ofBSA, BSA-2 .and BSA-1 in the BSA-1 mass spectra were obtained at higher laser energy (2.8 pJ/pulse) than that the mass corresponded to an average of5 different mass spectra on a same target. averaging of 256 lasers short (external calibration using horse skeletal muscle
region ofthe molecular ion peak. I!&\-2 and ofthe BSA mass spectrum (2.1 pJ/pulse). ~11 A mass spectrum was obtained with .s signal apomyoglobin).
Figure 1. Spectre dr masse MALDI de BSA, USA-2 ct BSA--1 montrant la region des ions molt?ulaires. Les specrrea de BSA-2 ec BSA-1 ont et& obtenus pour une Cnergie du pulse laser (2,8 pJ/pulse) plus GlevCe que celle ut.ilis& pour le specrre de BSA (2,l pJ/pulsc). Les spectres montrCs r&ultrnt d‘une moyenne de cinq spectres de masse rt?alis& sur un mCme &hantillon. Chaquc spectre de masse r&ulte du signal moyrn nbtenu j partir de I’irradiation de I’Cchantillon par 25(, tils laser.
38
Mass
bution of each conjugate to the stimulate peak. All these calculations were performed using the sofrware KaleidaGraph (version 3.0.2). As shown injgure2(a) a gaussianfit can be usedto satisfactorily describe the BSA experimental peak, except in the higher-mass range. Under the above assumption, the peak of BSA with n attached haptens should be gaussianand shifted by n X m (m = 312.2 Da for BSA-2 and m = 358.2 Da for BSA-l), the intensity of this peak being proportional to the relative abundance a(n) of,BSA with n attached haptens. The distribution a(n) was adjusted to get the best fit of the experimental peak. Such a procedure was recently applied by Tang et al. [9] to MALDI quantification of P-2-microglobulin glycosylated end products in human serum. Figure 2(b) and (c) respectively show the best simulation for the BSA-2 peak and the contribution of the different BSA-2 species.Only the most abundant species have been shown in figure 2(c). For BSA-2, the distribution of 2 extracted from this simulation is quite well described by a gaussian one centered on no = 10.5 molecules of 2/BSA molecule (mean square deviation (5()* = 8.2). Obviously, n,, is equal to the mean number of conjugates deduced from the experimental mass shift between BSA and BSA-2.
spectrometry
of protein-hapten
conjugates
Figure 3(a) shows a simulation of the BSA-1 peak using the distribution a(n) previously determined for BSA-2. The simulated peak does not fit correctly the experimental peak since it is shifted toward higher mass. This could indicate that the transformation of BSA2 to BSA-I is not complete. Another possibility would be to consider a lactonization of the hapten in the acidic matrix solution (schemeli. F’gure 3(b) and (c) show the best fits respectively for BSA-1 and the lactone form of BSA1. In both cases,the hapten distributions were gaussianand centered respectively on no = 10.0 (oo’ = 8.7) and tie =10.2 (o,,’ = 8.7). These values are closeenough, taking into account the experimental uncertainties, to conclude that: BSA-2 has been transformed almost completely into hapten, showing that this simulation approach can be very useful to follow a chemical reaction carried out on a conjugate.
The hapten quantification was also achieved with a radioactive tracer [14C]-l. After exrensive dialysis to free the conjugate from the excessKCN, the remaining radioactivity on the protein was counted. This method gives 9 * 1 molecules of l/BSA molecule [3] in good agreement with the MALDI results. This good agreement shows that the assumption used for the simulation is quite reasonableand that all 1 lo2
1on
r
60
65000
66000
67000
68000
69000
70000
64000
nla*s
66000
68000
70000 72000 nlass
74000
76000
781100
Figure 2. (a) Experimental BSA peak (-) and simulation (- - -) by a gaussian distribution. (b). Experimental BSA-2 peak (-) and the best simulation (- - -) using a gaur.sian distribution of2 (see text). (c). Details of the hapten diatribution for BSA-2. The upper curve corresponds to the sum of all the BSA-2 contributions.
640M)
66000
68000
70000 72000 ln‘lss
74000
76000
78000
Figure simule masse lation (- -) pour toutes
2. (a) Pit de masse exp&imenral de BSA (-) et pit par one distribution Sausienne (- -). (b) Pit de exp&imental de BSA-2 (-) et la meilleure simuobtenue B partir d‘une distribution gausienne de 2 (voir texts). (c) D&ails de la distribution d’hapt&cy RSA-2. I a courbe sup&ieure indique la somme de Its contributions de BSA-2.
N. Gourlaouih
et al.
80 60
68000
72000
76000
mass
68000
64000
72000 mass
76000
Figure 3. (a) Experimental
8 10’ no=10.2
olP=n.7
f!! 610’
x .2
BSA-1 peak (-) and simulation (- -) uring the hapten distriburion found for BSA2 (see rext andjgureZ(c)). (b) Experimental BSA-1 peak I -) and the besr simulation (---) (see rexr). (c) Experimental BSA-I peak (--) and rhe besr simulation (- - -) assuming a complete lacronizarion of BSA-1 (see rexlj.
Figure 3. (a) Pit de masse exp6rimencal
2z 4 10' IE 2 10’ 0 lu” 64000
68000
72000 mass
76000
the factors implied in dze mass centroid and the width of the BSA peak (matrix and/or cat-ion adducts, ion initial velocity distribution, metastable decomposirion 171) are nor srrongiy modified when hapten is attached. Further experiments on other protein conjugates including the comparison between different techniques (radioactivity, ESI-MS) would be very useful to further validate the MALI31 approach. This technique could be quite general and
applied aswell for instance ta the srudy of rhe distribution
of post-translational
modification.
simulation pour BSA-2 p6rimental obrenue (-BSA-1 (-) posanr une
de BShl (-) er (-- -) utilisanr la disrribution d’haptenes trouv& (voir texte erfigun ,? (c)). (b) Pit de masse exde BSA-1 (-) et la meilleure simularion -) (voir rexrel. (c) Pit de masse exp&imcntal de er la meilleure simularion obtenue (- -) en suplacronisacion complPre de BSA-1 (voir texrc).
[3] (a) Gourlaouen Paris, 1995. (b) Gourlaouen ted.
(b) Harrman (1394)
(b) Pierce J., Andrew (1986)
Chem
40
D., Marquer G.H.,
M.R.,
Ann.
Ann.
A.. ,ubmitRev. Bwchem.
Rev. Biochem.
T.J., Pierce 1.. Schlws B 313 (1986) 397.
T.J., Lorimer
Jaworowski A., Hartman Chcm. 259 (1984) 6783.
CC)
[6] I’iercc
J., Toolbert 934.
[81 Beavis R.C.,
63
G.H.,
J.V.,
1. Biol. Chern.
N.E.,
F.C.,
Barker
lttst
I.A.,
I. Riol.
R., Biochemistry
A., Karas M., Hillekamp
19
F.. Giessman IJ., lnt. .$45-354.
Spsctrom. Ion Processes 1.11 (1994) Chair
B.T., Anal. Chrm.
62 (1990)
1836
1840. [91 Tang X., Sadeghi
P.C., Biowniuga:arc
P-et-M.-Curie,
10248.
[I] AdamcLyk M., Buko A., Chen Y.Y.. Fishpaugh J.R., Gebler J.C., Johnson D.D., Rioconjugate Chcm. i M., Gehler J.C.. Mattingly 7 (I w6) 475-481
EC., Harpel
G.H., Andrew Phil. Trans. R. Sot. Land.
171 Ingendoh J. Mass
631-635.
Lorimcr
[51 (a) Lorimer
261
University
197.
References
(1794)
Thesis
N., Florentin
141 (a) Mizmrko H.M., 52 (1983) 507.
(1980)
[2] AdamcLyk
N.,
Mcllwain 3740-3745.
L.K.,
M., Olumec Z., Verres A., Rraatz J.A., Dreifuss P., Anal. Chem. 68 119%)