Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric characterization of high molecular mass Starburst™ dendrimers

E LS EVI E R

International

Journal of Mass Spectrometry and Ion Processes 165/166 (1997) 405-418

Moss spootromotlv and Ion Recesses

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric characterization of high molecular mass StarburstTM dendrimers Ljiljana PaSa ToliC”, Gordon A. Andersona, Richard D. Smith”?“, Herbert M. Brothers IIb, Ralph Spindlerb, Donald A. Tomaliab “Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Mail Stop P8-19, Richland, WA 99352, USA bMichigan Molecular Institute, Midland, MI 48640, USA

Received 10 April 1997; accepted 4 June 1997

Abstract A series of StarburstTM polyamidoamine (PAMAM) dendrimers, built from a tetrafunctional ethylenediamine (EDA) core, generations 1 to 10 (Gl-GlO), and covering a wide range of charge state distributions (i.e. from m/z - 500 to m/z - 15 OOO), have been investigated by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (IT-ICR) mass spectrometry. High resolution mass spectra for lower generation dendrimers (Gl, M, - 1429 u to G5, M, - 28825 u) allowed characterization of the polydispersity of these systems, providing a basis for improving synthetic strategies and characterizing surface-modified dendrimers. The relationship between charge state in the gas phase and molecular mass of these spherical macromolecules is reported and used to estimate the stoichiometry of a G5 PAMAM dendrimer - polyclonal rabbit IgG immunoconjugate. The results presented herein represent the highest molecular mass species yet detected by FT-ICR MS with the exception of measurements based upon individual ion detection. 0 1997 Elsevier Science B.V. Keywords:

fl-ICR;

Electrospray

ionization;

Dendrimers;

1. Introduction

Starburst PAh4AM dendrimers belong to the recently defined fourth class of macromolecular

*Corresponding author. e-mail: rd _ [email protected] 0168-1176/97/$17.00

Tel.:

+ 1

504

3760723;

Polymers; Antibodies

architecture known as dendritic polymers [la,lb]. As a result, dendrimers have many unique structural and physical properties [2]. Starburst PAMAM dendrimers are synthesized in successive ‘generations’, each with a defined size, shape and surface chemistry, and possess the following distinguishing architectural features: an initiatorcore region, an interior region composed of re-

0 1997 Elsevier Science B.V. All rights reserved.

PI2 SO168-1176(97)00161-4

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peating branch cells radially connected to the initiator-core, and an exterior (surface) region of terminal units which comprise the outermost generation. The PAMAM family in this study is derived from an ethylenediamine (EDA) initiatorcore and an amidoamine repeating unit (Fig. 1). Because of their well-defined characteristics and unique properties, Starburst dendrimers are finding utility in a variety of applications, many biological in nature [3a-3i]. These spherical macromolecules can mimic certain properties of micelles, liposomes, and even large biomolecules to act as drug carriers or immunogenes. An important feature of PAMAM dendrimers is that a number of different molecules can be conjugated to the surface groups of the dendrimers. After attachment of antibodies or other targeting moieties, dendrimer conjugates have been used to deliver a large number of boron atoms for neutron capture therapy [3d], act as linkers for the

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attachment of radioisotopes to the surface of the dendrimer [3a], and replace a secondary antibody in an improved diagnostic assay [3c,3i]. Dendrimers conjugated to chelating groups perform as highly efficient contrast agents for magnetic resonance imaging which could lead to a new class of blood pool contrast agents [3e]. Sugar moieties conjugated to the surface groups of a number of different dendrimer families have shown an enhanced ability to modulate the agglutination of influenza viruses [3f]. Finally, PAMAM dendrimers are showing utility as synthetic vectors for the delivery of genetic materials into a variety of cell lines [3g,3h]. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) [4-61 coupled with electrospray ionization (ES11 [7] has been shown to greatly extend the capabilities of mass spectrometry for the characterization of macromolecules [Sa-8i]. Studies of larger proteins

(a)

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b) WV---~~

HN-l

2

2 2

H*N-++N

HzN \

N-N f

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.,NHz

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‘NH2

H2N. HzN x

I/

GENERATION

1

GENERATION

2

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i; HzN

GENERATION

GENERATION

0

(2+N)

Fig. 1. Schematic diagram of the synthesis of ethylenediamine (EDA) core Starburst PAMAM dendrimers (top), and schematic diagram illustrating various defects which may lead to divergence from ideal dendrimer development (bottom).

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407

(b)

NRz

Missing Repeat Unit 0

0

N /\/

NH2

Intramolecular Cyclization

H

Cw.-60)~

NH-

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0 Fig. 1 Continued.

and/or biopolymer complexes typically require measurements at relatively high m/z. Although IT-ICR MS offers promise for analyses at high m/z, the achievable performance (i.e. high mass resolution and precision) significantly decreases with increasing m/z; thus, such studies are currently problematic [91. The experimentally practical upper m/z limit is often significantly lower than the predicted ‘critical m/z’ (i.e. the highest m/z that can be trapped for given experimental conditions) [5], due mostly to the imperfections of the cell geometry and of electrostatic fields associated with ion trapping and cyclotron excitation. Additionally, the injection and trapping of high m/t ions with a broad range of ion kinetic energies typically requires the use of high trapping potentials, and consequently results in a lower radial critical m /z, since the radial compo-

nent of the electric field effectively reduces m/z range of an ICR trap. The ion charge state and the Coulombic interactions between ion clouds of adjacent isotopic species in the ICR trap determine the upper molecular mass limit for unit resolution (M,,,,,), and consequently for the generation of high accuracy mass measurements. For typical operating conditions and trap parameters, the upper mass limit for which isotopic resolution can be obtained has been estimated to be M,,,,ar N 104B, where B is the magnetic field strength [lo]. However, it is well documented that ES1 can and that this can produce ions having high m/t, often be attributed to the generation of ‘compact’ structures (e.g. ionization under gentle non-denaturing conditions for proteins). Low-charge state non-covalent complexes (e.g. multimeric proteins) [lla-lld,12a-12f] have been reported, consistent

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with their compact structure and expected Coulombic constrain upon maximum charging. Thus, in order to improve applicability of FT-ICR MS to such biologically important systems, it is necessary to extend both the accessible mass and m/z range, and to more effectively realize the impressive capabilities of FT-ICR MS in the lower m/z range. The characterization of Starburst dendrimers (i.e. elemental composition, molecular mass vs. generation, homogeneity, interior and exterior groups, structure and dimension) has been accomplished by the use of common techniques (e.g. low-angle laser light scattering (LALLS), size exclusion chromatography, vapor-phase osmometry, intrinsic viscosity measurements, electron microscopy, IR, NMR and mass spectroscopy). Many of these analyses become increasingly problematic for higher generations. The use of matrixassisted laser desorption/ionization (MALDI) and FT-ICR MS has been reported for characterization of dendrimers (M, < 1.5000 u) resulting from an exponentially accelerated convergent synthetic approach [13a,13b]. The same class of dendritic molecules (kf, < 50 000 u) has been analyzed using MALDI time-of-flight (TOF) MS [13a-13c]. Although these analyses proved challenging (i.e. matrix incompatibility, because phenyl acetylene dendrimers are soluble only in nonpolar solvents), they have resolved defect structures (i.e. incomplete couplings), indistinguishable by size-exclusion chromatography and/or ‘HNMR. ESI-MS analyses of Starburst dendrimers have been previously reported, including molecular mass and polydispersity index determinations for a G4 PAMAM dendrimer (M, - 10 600 u) [14]. Low resolution mass spectra for a series of PAMAM dendrimers, generations 1 to 10 (Gl-GlO), have been previously obtained at our laboratory using a triple-quadrupole instrument (for Gl-G3) and a low resolution extended m/t range quadrupole mass spectrometer (for G4-GlO) [15]. Based on these data, a good linear relationship was obtained between Mrz/3 and the experimental number of charges (M,/(m /z)), consistent with the theoretical models predicting maximum charging controlled by Coulombic effects [16].

(1997) 405-418

ES1 coupled with IT-ICR offers a potentially attractive approach for the characterization of macromolecules. Herein, we report the detection of high molecular mass (up to N 1000000 u) Starburst polyamidoamine (PAMAM) dendrimers in an ICR trap, and compare them with data published earlier. High resolution and mass measurement accuracy, obtained for lower generations (i.e. Gl-G5), provided accurate molecular masses for the various components present in complex synthetic material. These data may also be used for determination of polydispersity values for these synthetic polymer systems. We also report initial attempts to characterize a conjugate of polyclonal rabbit IgG with G5 PAMAM dendrimer, an application that highlights the current limitations of IT-ICR technology.

2. Experimental

EDA-core Starburst PAMAM dendrimers, Gl-GlO, were synthesized in a stepwise process as described previously [2]. The G5 PAMAM dendrimer-immunoconjugate was prepared by treating 1 mg of polyclonal rabbit IgG (rIgG, dissolved in 750 ~1 of phosphate buffered saline (PBS) of pH 7.4) with 300 ~1 of 0.1 M NaIO,. After a 30-min incubation, the rIgG was purified on a PD-10 column with PBS as the eluent. The purified protein was then treated with 1 mg of G5 PAMAM dendrimer. After 1 h at ambient temperature, 100 ~1 of a 4 mg/ml solution of NaBH,CN in PBS was added. After an overnight incubation at 4°C the reaction mixture was concentrated on a Centricon 30 microconcentrator knicon), and the conjugate was isolated by ion exchange chromatography. Starburst PAMAM dendrimers Gl-GlO were electrosprayed from 5% HOAc or 10 mM NH,OAc solutions, in the concentration range 0.1-l mg/ml. We found that the solution conditions did not significantly influence the spectra. The previously reported PAMAM dendrimers spectra were obtained from 1 mg/ml solutions in 10 mM NH,OAc (pH 8.6) with 10% MeOH, on a

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Finnigan triple quadrupole (Gl-G3) and an extended m/z range quadrupole (G4-GlO) mass spectrometers [15]. Polyclonal rabbit IgG (1 mg/ml) and polyclonal rIgG-G5 PAMAM dendrimer conjugate (400 pg/ml) were desalted by the use of an off-line microdialysis procedure, described elsewhere [ 171, and were electrosprayed from 10 mM NH,OAc solution. The 7-T ES1 FT-ICR mass spectrometer has been described elsewhere [8a], as well as a custom ES1 source and interface incorporating an rfquadrupole for collisional focusing (Pass Tolic et al., unpublished results). Briefly, the ES1 source consists of a heated stainless steel ‘desolvation’ inlet capillary, a l-mm orifice diameter skimmer, and a short quadrupole segment added to the set of two quadrupole ion guides used in the original configuration and operated in rf-only mode ( N 750 kHz, N 500 VP,). Mass spectra were obtained utilizing standard experimental sequences employing broadband (BB) or single frequency (SF) selective ion accumulation (SIA) using quadrupolar excitation (QE) [18a-18c]. Colored noise QE waveforms, generated by use of a PC board (PCIP-AWFG, 5 MHz, 12 bit, Keithley Metrabyte Co., CA> were sequentially repeated during QE event, and typically involved 20 Vpp and 1 Vpp quadrupolar fields for BBQE (over N 3000 m/z units) and SFQE, respectively. Ion accumulation was accomplished at N lop5 torr of N,, injected into the cell via a piezoelectric pulse valve (Lasertechniques Inc., Albuquerque, NM). For higher generation dendrimers (i.e. G6-GlO), data were acquired with QE waveforms covering gradually increasing m /z ranges, accompanied with gradually increasing trapping voltages, and summing resulting spectra, in order to compensate for different kinetic energies of species with significantly different m/z values. An Odyssey data station (Finnigan, Madison, WI) provided ICR trap control, data acquisition and storage. The signal to noise for the high resolution G5 PAMAM dendrimer mass spectrum, was enhanced by zeroing [19,20] or inverse apodizing (Welch) of the noise between beats prior to Fourier transformation, using our own custom data analysis software, ICR-2LS [21].

409

3. Results and discussion

Isotopically resolved ES1 FT-ICR mass spectra obtained for Gl-G4 PAMAM dendrimers are summarized in Fig. 2. In all cases, a cluster of peaks was observed that included peaks corresponding to the theoretical mass of the molecular ion with the structure of a perfect dendrimer (see Fig. 1); these peaks, however, did not necessarily represent the most significant (i.e. the highest abundance) species. A large number of species are evident in these samples resulting from incomplete monomer addition and/or intramolecular cyclization during synthesis (Fig. 11, and the number of identifiable components increases with each successive generation. The alternative forms of dendrimers include species differing from the perfect polymer by integer numbers of polyamidoamine repeat units (114 u), or species corresponding to loss of an ethylenediamine unit (intramolecular cyclization), and thus differing by 60 u from the complete or truncated polymers. In previously reported low resolution ES1 mass spectra, the charge state distributions corresponding to G4 and G5 PAMAM dendrimers were barely resolved and unresolved, respectively, thus making it difficult to obtain molecular mass (M,) information for dendrimers with M, > 10000 u. The high resolution spectrum of a G4 PAMAM dendrimer (Fig. 2) shows prominent isotopically resolved signals corresponding to MI,.+, = 14 214.4 u, in good agreement with predicted Mr,th = 14213.9 u (masses reported for Gl-G5 PAMAM dendrimers correspond to the mass of the most abundant isotopic species, while the values reported for G6-GlO correspond to average molecular masses). Significantly greater heterogeneity relative to the smaller dendrimers, primarily due to reaction incompleteness is evident in this spectrum. Polydispersity, defined as the ratio of weight average (M,) to number average CM,,) molecular mass, is usually determined by the use of size exclusion chromatography, ultracentrifugation, LALLS, or intrinsic viscosity measurements. It is possible that a more precise appraisal of polydispersities may be made by the use of MS. Kallos et

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zt.,[,.l

(1997) 405-418

G2

3l+

M,,t,,=3,255.3 u M,,,,=3,255.5 u

+ 4

63

7+ 6+ 5+

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4+

500

1000

1500

2000

2500&ObO

3600

4060

4800

sob0

64

1485

1585

Polydispersity (Mg4J=1.0003

500

1000

1500

2000

3bOO 000 ld

3soo

4000

45006000

Fig. 2. ES1 FT-ICR mass spectra of Starburst PAMAM dendrimers generations 1 to 4 (Gl-G4). All spectra show a relatively high abundance of dendrimers with defective structures, resulting from different combinations of various numbers of missing amidoamine units, A = 114 u (i.e. branching defects), and/or ethylenediamine units, A = 60 u (i.e. intramolecular looping). Inserts in the Gl spectrum show dimeric species [(M - 60) + 2HlZ+ and illustrate the typical low abundance of interlinked structures in the analyzed lower generation dendrimers. Inserts in the G4 spectrum show m/z-expanded regions for (M + 7H)‘+ ions for ideal and truncated and/or bridged forms of G4, used to determine polydispersity index (i.e. M,,,/M, = [(~niM~i/zniM,,i)/(~n~~~,i/zni)] = 1.0003), and comparison of experimental (Mr,th) isotopic distributions for (M + 7H)‘+ G4 ion with (Mr,exp ) and theoretical perfect structure.

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al. [14] calculated the polydispersity value of an ammonia core G4 PAMAM dendrimer, M, * 10600 u, to be 1.0007, based on the mass spectra obtained with ESI/triple quadrupole instrumentation. Our data give the value of 1.0003 as polydispersity index of an EDA-core G4 PAMAM dendrimer, M, _ 14214 u, that compares favorably with polydispersity indices determined for similar systems. More importantly, high resolution FT-ICR mass spectra allowed significantly better analysis of the sources of polydispersity, i.e. overlapping peaks, resulting from different combinations of various numbers of missing amidoamine (due to the branching defects) and/or ethylenediamine (due to the intramolecular looping) units, are clearly resolved (Fig. 2, bottom). These spectra also indicate that analyzed lower generations do not contain a significant contribution of interlinked structures, that are the result of different intermolecular bridging or looping processes, and are generally associated with increased polydispersity (see Fig. 2 top). The ESI-FT-ICR mass spectrum of a G5 PAMAM dendrimer obtained by the use of BBQE (Fig. 3 top), resolved the charge state distribution centered at _ m /z = 2350 u; however, isotopic resolution was not obtained. High resolution spectra were obtained by the use of SFQE, i.e. quadrupolar excitation at the reduced ICR frequency of (M + 12H)12+ species (Fig. 3 bottom), resolving contributions from a large number of species, separated by 60 u or 114 u. A signal corresponding to the ideal structure of an EDAcore G5 PAMAM dendrimer, has been identified (by overlapping experimental isotopic distribution with the theoretical isotopic envelope, Mr,th N 28825 u) at Mr,_ N 28821 u, although heavily buried in the cluster centered N m/z - 2402. Time domain data sampling, used to generate the spectrum shown in Fig. 3B, involved inverse (Welsh) apodization of the data between the ‘beats’, in contrast to the previously described approach of setting all data between the beats to zero-amplitude [19,20]. As it is well known, the abrupt truncation of a time-domain signal results in Gibbs oscillations in the frequency-domain spectrum; these are normally reduced by applying any of several windowing functions before Fourier transformation (i.e. apodization). Similarly, re-

411

placement of partial signal zeroing by inverse apodization reduces these artifacts, while the spectral quality is still significantly improved. This spectrum illustrates the advantage of high-resolution routinely achievable with FT-ICR instrumentation in the characterization of complex synthetic polymers. High resolution spectrum allows M, determination with significantly higher accuracy (i.e. 28850 u from low-resolution spectrum vs. 28821 u from high resolution spectrum), and more detailed characterization of Starburst PAMAM dendrimers, and consequently the potential for improvements in synthetic strategies. ESI-IT-ICR mass spectra of higher molecular mass dendrimers (G6-GlO) become progressively more complicated due to the propagation of errors introduced in earlier generations. The resulting synthetic material has greater heterogeneity that ultimately results in an overlap of different charge states. In combination with high M, [lo], the spectra become unresolvable using current instrumentation. Note that this ‘cut-off of isotopically resolved FT-ICR mass spectral data (i.e. the necessity for charge state and, consequently, M, determination) is significantly higher than for charge state resolved quadrupole mass spectral data (i.e. G5 vs. G3). Mass spectra obtained for G6-GlO are shown in Fig. 4, and illustrate the gradual shift to the higher m/z values with increased M,. The spectra for G6 show a broader and apparently bimodal distribution, i.e. two broad peaks, one of which overlaps with rather narrow charge state distribution obtained for G7, and that is also prominent in the extended m /z range quadrupole spectrum reported earlier [15]. The charge state distributions for G7, and G8 span relatively narrow m /.z ranges, and do not overlap with distributions obtained for next (higher) or previous (lower) generation, indicating effective syntheses. We have previously attributed the observation of ‘narrow’ charge state distributions to systems having low ‘structural heterogeneity’ 1151;the present results are completely consistent with this hypothesis. The samples are not from the same genealogical family, which explains the relatively low structural heterogeneity of G6 in comparison with G7 and G8. Spectra obtained for G9 and GlO show much broader charge state distributions than previous generations, and per-

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(M+13H)13+

(M+l2H)‘2+ , ,

G5

15

(M+12H)12+

mh

M,,,,=28,825 u

A

1500

2OQO

2500 m/z

3600

3500

Fig. 3. ES1 IT-ICR mass spectra of Starburst PAMAM G5 dendrimer: low-resolution spectrum (top) obtained by the use of BBQE (over 500 < m/z < 3500 range) showing resolved charge states for this highly heterogeneous material, and high-resolution spectrum (bottom) obtained by the use of SFQE (at m/z = 2400, corresponding to (M + 12H)12+ species). Insert shows m/z-expanded region for (M + 12H)t2 + ions for ideal ( Mr,exp) and truncated and/or bridged forms of G.5, with time domain data sampling prior to IT (see text), aligned with theoretical isotopic envelope for G5 with perfect structure.

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413

G5 M r,th- 28,825 u 2

0

G6 M r,th- 57,999 u 2

0

G7 M r,th- 116,491 u 2

0

m/z

GS M r,th- 232,677 u

G9

2000

4000

6000

8000

Fig. 4. ES1 FT-ICR mass spectra of Starburst PAhUM higher m /z values with increased M,.

10000&12000

14000 16000

l$OOO 20000

dendrimers generation 5 to 10, G5-GlO, illustrating the gradual shift to

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0

200,000

400,000

600,000

800,000

(1997) 405-418

1,000,000 1,200,000

1,400,000

Fig. 5. Least squares fitting to the function 4 =aMP; number of charges (q) were determined from ES1 FT-ICR mass spectra and plotted vs. theoretical molecular mass CM,,,,) for generation 1 to 10 Starburst PAMAM dendrimers (o maximum, and 0 average number of charges) and experimental results for proteins of known mass (W maximum and ? ?average number of charges), obtained by the use of ES1 FT-ICR or ES1 TOF mass spectrometers (see text). (q = Mr,th/(m/z))

haps indicates less successful syntheses and/or higher amount of impurities. An empirical relationship 4 = u&f:, b = 0.64 (for the maximum number of charges) or b = 0.58 (for the average number of charges), was obtained by least square fitting of the observed number of charges (4 = M,_/(m /z>) vs. theoretical molecular mass 0&h) (Fig. 5). The present results are consistent with previously obtained M, vs. number of charges behavior [15]; the slight shift to the higher number of charges is probably related to different solution conditions used (i.e. NH,OAc with 10% MeOH previously vs. 5% HOAc currently used), and possible impacts upon the latter stages of desolvation and related gas phase chemistry. Data acquired for the Starburst PAMAM dendrimers are compared with data obtained for some of the proteins electrosprayed under native

(i.e. physiologically relevant) conditions, including FT-ICR MS data obtained in our laboratory ((Nle4?4-oxalocrotonate tautomerase (40T), streptavidin, SecB, SecB/OppA, polyclonal rabbit IgG, Corynebactetiul sarcosine oxidase) and published ESI-TOF data (40T, streptavidin, catalases HP I and II, citrate synthase, immunoconjugate F(ab’)2-CPG2, soybean agglutinin, isolecitin) [12a-12fl. Although this set contains a significantly different class of molecules, the extent of charging produced with ES1 is quite similar to that observed for the dendrimers, strongly indicating that all of these species have compact structures. The slightly different empirical relation q = uMrb, b = 0.53 (for the maximum number of charges) or b = 0.54 (for the average number of charges), obtained in the case of proteins, can be ascribed to the inherent structural variations of these biopolymers, as well as the different

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(smaller) number of accessible (surface) protonation sites for different biomolecules. In this regard it should be noted that given sufficient high proton affinity charge states, proteins would be expected to display greater charging than the spherical dendrimer molecules of the same M, since any deviation from spherical shape would increase the number of possible charges. The fact that this is not the case can be ascribed to the much more limited number of likely charge sites on the protein surface and the fact that these sites will not be evenly distributed. As already indicated, solution conditions and subsequent gas phase processes might be expected to result in some (small) variation of gas phase charging by ESI. The extrapolation of the q vs. M, plot to larger molecular masses allows an estimate of m/z range in which electrosprayed ions of macromolecules should be expected, as shown previously [12a,15]. Starburst dendrimers are finding utility in a variety of biological applications, and Starburst dendrimer-(monoclonal/polyclonal) antibody immunoconjugates have potentially interesting applications. However, the nature of these conjugates is still uncertain. For example, an EDA-core G5 PAMAM dendrimer conjugated with polyclonal rIgG may theoretically contain anywhere from one to five dendrimers per IgG, based on the conjugation challenge ratio (i.e. the number of moles of dendrimer in comparison to the number of moles of antibody used in the conjugation). Fig. 6 shows the positive ion ES1 FT-ICR mass spectrum of the GS/rIgG conjugate, together with the mass spectrum obtained for rIgG polyclonal immunoglobulin with nominal weight of 150000 u, and the mass spectrum of the G5 PAMAM dendrimer. As a result of the heterogeneity of the individual components (both G5 and polyclonal rIgG), only a broad signal centered around m/z - 7400 was observed for the conjugate. This value correlates well with the m/z value calculated from the empirical q vs. M, relationship (protein data from Fig. 5) for a conjugate containing three dendrimers per rIgG molecule (i.e. (m/~),,,~, 7400). However, taking into account the high degree of heterogeneity and adduction observed for both partners, i.e. G5 and rIgG, there is a high

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415

probability that the charge distribution for the immunoconjugate will be shifted to somewhat higher values. Thus, it is more likely that the stoichiometry of the immunoconjugate is 2:1, i.e. two G5 molecules per single rIgG molecule, appearing at higher average m/z value (i.e. (m / hW,,,p - 7400) than predicted based on q vs. M, relationship (i.e. Cm /z),,~,, - 6900). Obviously, this conclusion is preliminary and quite speculative at this point. Nonetheless, ES1 FT-ICR data do agree with predicted stoichiometry, i.e. one to five dendrimers per IgG, corresponding to the average charge state distribution centered between Cm /z),,~, - 6500 (1:l immunoconju- 8200 (1:5 immunoconjugate) and (m/z),,,,, gate). These results also indicate that the use of monoclonal IgG would lead to more readily interpreted results. The fact that IgG can be effectively trapped and detected clearly suggests that non-covalent complexes of IgG with other species might be detectable; if this is the case, the methods of bio-affinity characterization mass spectrometry (BACMS) [22] would offer exciting new applications of IT-ICR MS. 4. Conclusions Starburst PAMAM dendrimers (Gl, ZL4,- 1429 u to GlO, M, - 934701 u) were successfully analyzed by the use of ES1 interfaced with a 7-T FT-ICR mass spectrometer. These results represent the highest molecular mass species yet detected by FT-ICR MS, with the exception of results based upon individual ion detection [23a-23c]. FT-ICR MS results were compared with mass spectra obtained earlier with the same samples in our laboratory by the use of triplequadrupole or extended m/z range quadrupole mass spectrometers. High resolution mass spectra obtained for lower generation dendrimers (Gl; M, - 1429-G5; M, - 28825) may be used to determine their polydispersities, e.g. a value of 1.0003 was obtained for an EDA-core G4 PAMAM dendrimer, M, - 14214 u. The increase in the molecular mass for each successive generation results in a shift of the observed charge state distributions to higher m/z values, resulting in a range of > 10000 m/z units spanned by these

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Noise

M r,th- 150,000 u M r,exp= 151,600 2500 u

GS/rIgG Conjugate

1,000

3,250

52;”

1,750

10,000

Fig. 6. ES1 m-ICR mass spectra for Starburst PAMAM GS dendrimer (top), polyclonal rabbit IgG (middle), and PAMAM GS-rIgG immunoconjugate (bottom). The stoichiometry of the immunoconjugate is estimated to be 2:1, i.e. two GS molecules per single rIgG molecule, on the basis of empirical q vs. M, relationships (see text).

10 generations. An empirical relation q = aM), b = 0.64 (for the maximum number of charges) or b = 0.58 (for the average number of charges), obtained by least square fitting of the observed vs. molecular number of charges (q = M&z/z>) mass (M,), is consistent with physical models predicting a compact (spherical) structure, with maximum charging governed predominantly by Coulombic effects. Such empirical relations may be used to qualitatively estimate the m/z range in which electrosprayed ions of large biopolymers should be expected, and allowed us to speculate concerning the stoichiometry of a G5 PAMAM dendrimer-polyclonal rabbit IgG immunoconjugate. Due to the heterogeneity of both the G5 and the polyclonal rIgG component, different charge states for the conjugate are unresolved using current instrumentation. However, based on the above-mentioned empirical relationships, we were able to estimate the most probable stoi-

chiometry of this immunoconjugate to be 2:l (i.e. two G5 molecules per single rIgG molecule). Finally, we note that advanced higher field (11.5 T) IT-ICR instrumentation that we are presently developing, offers the potential of resolving the complexity of higher generation dendrimers and the immunoconjugate species studied in this work. Acknowledgements We thank Drs. J.E. Bruce, M.V. Gorshkov and D.C. Muddiman for technical assistance and useful discussions on the subject of this paper. HMB, RS, and DAT thank Dendritech, Inc. for its support. This work was supported by a grant from the National Institute of Health (GM 53558) to Pacific Northwest National Laboratory (PNNL). PNNL is a multiprogram national laboratory operated by Battelle Memorial Institute for the US Department of Energy under Contract DEAC06-76RL0 1830.

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