Mass spectrometric approaches to molecular characterization of protein-nucleic acid interactions

Toxicology Letters ELSEVIER

Toxicology

Letters

82/83

(1995)

567-575

Mass spectrometric approaches to molecular characterization of protein-nucleic acid interactions Michael Przybylski”‘“, Jiirgen KasC, Michael 0. Glockera, Eberhard Hans R. Bosshardb, Steffen Neck”, Mathias Sprinzl’

Di_irraTb,

“Faculty of Chemistry, University of Konstanz, P.O. Box 556OM731, 78434 Konstanz, Germany bBiochenlical Institute, University of Zurich, Winterthurer Strasse 190, 8057 Zurich, Switzerland ‘institute of Biochemistry, University of Bayreuth. P.O. Box 101251, 9.5412 Bayreuth. Germany

Abstract The recent development of ‘soft’ ionization-desorption methods has lead to a breakthrough for the mass spectrometric analysis of biomacromolecules such as proteins and nucleic acids. In particular, the feasibility of electrospray-ionization mass spectrometry (ESI-MS) for the direct characterization of non-covalent supramolecular complexes is opening new analytical perspectives. Examples hitherto analyzed by ESI-MS include enzyme-substrate and -inhibitor complexes, homo- and heterodimers/ trimers of leucine zipper polypeptides, and several other DNAand RNA-binding proteins. Furthermore, the characterization of double-stranded and higher-order oligo- and polynucleotide complexes by negative-ion ES1 has been demonstrated. Ions specific of non-covalent protein and oligonucleotide complexes can be selectively dissociated by changing the solution conditions and by increasing the desolvation potential. These results form the basis for the molecular characterization of protein-nucleotide interactions, thus complementing protein-chemical approaches, and other methods of structure determination. Keywords: Supramolecular protein complexes; Electrospray-ionization mass spectrometry; peptides; Double-stranded oligonucleotides; Protein-nucleotide interactions

1. Introduction The direct mass spectromettic analysis of biomacromolecules has experienced a breakthrough in recent years by the development of efficient ‘soft’ desorption ionization methods such as fast atom bombardment (FABMS), 252-Cf-plasma desorption (PDMS), and particularly, electrospray-ionization (ESI-MS) and matrix-assisted

* Corresponding

author.

037%4274/95/$09.50 @ 1995 Elsevier SSDI 0378-4274(95)03502-C

Science

Ireland

Leucine zipper poly-

laser desorption-ionization (MALDI-MS) [l]. Intact biopolymer molecular ions have been obtained most successfully by ESI- and MALDIMS (up to several 100 kDa for proteins) [2,3]. In combination with specific chemical or mass spectrometric fragmentation, these methods have found already broad application to primary structure characterization such as sequence determinations and covalent post-translational structure modifications [4]. In contrast to MALDI-MS which produces essentially denatured protein molecular ions, multiple charged molecular ion series and distributions of ‘native’ protein

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M. Przybylski et al. I Toxtcology Letters 82183 (1995) 567-575

568

states in solution are obtained by ESI-MS (e.g. [M + nH]“+), and have been shown to provide information about solution conformation and tertiary structures [2,5]. Moreover, the recently discovered feasibility of ESI-MS in the direct analysis of intact supramolecular complexes is leading to new analytical perspectives, particularly the study of non-covalent protein interactions [5,6]. A number of specific non-covalent complexes of biopolymers have been analyzed by ESI-MS already, such as enzyme-substrate or -inhibitor complexes, protein quarternary structures and double-stranded oligonucleotides [6-111. These successful initial model studies have prompted our interest and provide the basis for a systematic evaluation of analytical preconditions and applications of the ESI-method to the characterization of protein-polynucleotide interactions. In this article, recent studies in our laboratory on (1) leucine zipper polypeptides and other DNA- and RNA-binding proteins, and (2) protein- and nucleotide-interactions of the elongation factor-Tu (EF-Tu) from T. thermophilus are summarized. The current results indicate ESI-MS as an efficient tool for the direct identification of protein-nucleotide interactions at the molecular level, hence extending and chemical and spectroscopic complementing methods for their structural characterization. 2. Results and discussion 2.1. Macromolecular ion formation and preconditions for analysis of non-covalent protein complexes by ES-MS First successful analyses of non-covalent complexes by ESI-MS have led to an increasing number of model studies, and applications to Table 1 Preconditions Criteria

and ESI-MS

for identification

parameters

for identification

of supramolecular

+ Specific complex stoichiometry/equilibrium components + Competition of complex components + Specificity of solution conditions + Gas-phase stability of complex ions

complex of complex

supramolecular biopolymer structures as well as synthetic self-assembly systems [5,12]. Furthermore, this unexpected feature of the electrospray method has stimulated the detailed evaluation of the ion formation mechanism(s) by field-induced desolvation from solution [13]. Although the pathways of desolvation and macromolecular ion-formation have not yet been elucidated sufficiently well to derive direct information about tertiary structures, useful correlations between solution structures and multiple charged ions [7,10] have been obtained. A qualitative differentiation of charge state distributions (‘charge structures’ [5]) has been observed for ‘native’ protein structures compared to spectra of irreversibly denatured proteins, e.g. upon thermal and solvent-induced denaturation [7]. An illustrative example demonstrating the correlation of solution structure and molecular ion (charge) pattern has been the pH-dependent dissociation and reconstitution of myoglobin, one of the first noncovalent protein complexes detected by ESI-MS [6]. Dissociation by acidification yielded characteristic high charge-state ions of the apoprotein, whereas renaturation at pH 6 provided the reconstituted intact heme-protein ions [lo]. Furthermore, structural states of proteins have been probed by deuterium exchange in solution and by comparative analyses of chemically modified proteins yielding results consistent with NMR data [7,14,15]. Although little is yet known to which extent solution structures are reflected by charge distributions of molecular ions these results appear to fulfil one prerequisite for detecting supramolecular protein complexes. Preconditions and parameters of ESI-MS which have been employed for the characterization of non-covalent protein complexes are summarized in Table 1. Most important is the evalua-

of non-covalent ESI-MS

nrotein

and polynucleotide

parameters/dissociation

complexes by

Increase of declustering voltage (-20-100 V). Increase of interface temperature. Change of solution stoichiometry. Chemical modification/specific mutation of components. Change of pH; buffer; concentration. Increased declustering voltage. Collision-induced drssociation.

569

M. Przybylski et al. I Toxicology Letters 8218.3 (1995) 567-575

tion of experimental conditions that provide (1) the differentiation of non-covalent complexes and covalent adducts, and (2) the distinction of possible unspecific aggregation products or ‘cluster’ ions. The latter artefact ions - although observed in most desorption-ionization MS methods [5] - do not appear to be significant under ESI-MS conditions [7]. A critical step for the detection of non-covalent interactions is the desolvation of macro-ions supported by a small declustering (ca. 10-100 V) potential between the inlet capillary tip and skimmer (repeller) electrode (counter-electrode-skimmer potential, ACS); the lower ACS, the higher is the chance that non-covalent complexes survive the desolvation process as illustrated by the observation of water-solvated polypeptide ions at ‘incomplete’ desolvation conditions [7]. A number of experimental parameters may be required to ascertain specific binding structures, such as competition studies of complex components or changing solution conditions (pH, concentration). Non-covalent protein complexes analyzed under these conditions encompass different types of ligands and binding (such as ionic and hydrogen-bond interactions), with dissociation constants ranging from lop6 to lo-” M [6]. These model studies suggest broad application potential of ESI-MS for the study of protein- and nucleotide-interactions. 2.2. Identification of leucine zipper complexes Several leucine zipper classes of proteins have attracted interest in the last years because of their function in regulating transcription by specific DNA recognition [16]. The leucine zipper structural motif, consisting of a repeating 4-3 heptad of hydrophobic and non-polar residues with Leu dominating at position 4, was first detected in one of the simplest DNA-binding structures, the basic region leucine zipper (bzip) where it mediates the dimerization of 2 basic regions to a DNA-binding site [17]. Probably the best known leucine zipper is the C-terminal domain of the yeast transcription-activator, GCN4-pl which thus seems to be an appropriate model system for studying corresponding protein and nucleotide complexes by ESI-MS [18,19]. As

shown by the crystal structure, GCN4-pl assembles to a dimeric parallel coiled coil in which hydrophobic interactions between the ry-helical polypeptide chains are the dominant stabilizing forces (see structure in Fig. 1). Systematic studies by ESI-MS were carried out on a series of synthetic leucine zipper polypeptides, based on the sequences of naturally occurring coiled coils [16]. The ES1 spectrum of GCNCpl at low declustering potential (ACS, 10 V) is shown in Fig. la. The direct identification of a dimeric complex, (M2, M, 8078 Da) is provided by the 5 + charged macro-ion at m/z 1617 ((2 X 4039+5)/5 = 1616.7) whereas the ions at m/z 1347 and 1010 can originate from both the monomer and/or dimer (M)3’/(M2)6’,

AC-R MKaEDK Monomer

a

VEEOLSK

NY@NE

VA@KL

3+ (M)

IOV

b

a+ CM)

6OV

1347

1314?

4+ (M) 1010

VGER-CONH,

/ M, 4039

5+ Drmer 1616

I

_;,L.I

2000 mh

,.

4+(M) 1010

1000

i /

2000 m,z

Fig. 1. ES1 mass spectra of leucine zipper polypeptide GCN4-pl at 10 (a) and 60 V(b) repeller declustering voltage (ACS). The dimeric structure is only observed at ACS = 10 V by the 5 + charged molecular ion. Analysis was performed by injection of 5 ~1 50 PM peptide in 2 mM NH,OAc:methanol (9:1), pH 6.0. The top insert shows the coiled-coil GCN4-pl crystal structure, complexed with the complementary oligo-

nucleotide [17].

M. Przybylski

570

et al. I Toxicology

and (M)4’/(M2)8+, respectively. Generally, unequivocal identification of an oligomer composed of identical polypeptide chains is obtained by a molecular ion with a non-integer charge number, when divided by the number of complex components, i.e. charge of molecular ionlnumber of chains # integer number. Hence leucine zipper homodimers are characterized by all oddcharged ions. The peak of the [M + SH]” ion of the dimer completely disappears upon dissociation at higher ACS (60 V, Fig. lb) whereas the 3 + and 4 + charged ions of monomeric GCNCpl remain unchanged which confirms the non-covalent nature of the complex. Several model leucine zippers based on the sequences of natural coiled coils have been designed, one of which (‘coil-Ser’) has been crystallized recently and shown to form a 3stranded coiled coil [20]. In the ES1 mass spectrum of a synthetic peptide, LZ, whose sequence LZ: AC-E YEALEKK LAAWEAK LZILA. AC-E YEALEKK LAAHEAK a

Dlmer4’

Letters

82183 (1995) 567-575

is identical with that of coil-Ser except for Trp at position 2 and Ser at position 14, the formation of a trimer was identified by an (M3)5+ ion at m/z 1986 whereas no dimer (M2)5+ was observed [21]. The triple-stranded complex of LZ is in agreement with the crystal structure of ‘coilSer’ and was confirmed by sedimentation equilibrium analysis [21]. Moreover, direct information in equilibria between dimers and trimers and the formation of homo- and heteromeric complexes was obtained by ESI-MS in hybridization experiments of LZ peptides containing different sequence variants. As an example, Fig. 2 shows corresponding spectra of a mixture of LZ and LZ( 12A) containing an Ala-substitution in position 12 that destabilizes the coiled coil. Ions characteristic due to the heterodimer, AL4+, and both possible hetero-trimers (LZ)/(LZ12A), and (LZ),/LZ12A were identified, in addition to the homomeric complexes. In contrast to the low LQALEKK LEALEHG-CONH,

(L)

LQALEKK LEALEHG-CONHp (A) Dimers

Repeller 10 V

AL

1633 04

’ ,,LL A2’

AAL

!

ALL \ ,’

2000 Monomerz’

1654 06

II

AAy,

Repeller 20 V

m/z

1640

/, ,, m/z

1660

Trimers ALL5+

AAAL” ,L Turners+

1500

Fig. 2. ES1 mass spectra LZ12A. (a) Spectra of spectrum); (b) spectrum 4 + charged dimeric and

1960

1960

m/z

of dimeric and trimeric heteromers obtained from equal volumes of 0.1 mM solutions of peptides LZ and dimeric and trimeric complexes on a quadrupole mass spectrometer at ACS = 10 and 20 V (bottom of dimers (top) and trimers (bottom) on a magnetic sector instrument, showing isotopic resolution of 5 + charged trimeric ions.

M. Przybylski et al. I Toxicology Letters 82183 (1995) 567-57-T

resolution ES1 spectra obtained with a quadrupole mass analyzer (Fig. 2a), unequivocal assignment of dimeric and trimeric complex ions was achieved on a double focusing magnetic sector instrument that permitted isotopic resolution (Fig. 2b). Thus, a direct differentiation of the 4 + charged homodimer ion within the 2 + charged monomer was provided by the mass differences (Am, 0.25 amu) of isotopic multiplets at m/z 1633 and 1654. Likewise, correct isotopic resolution (Am, 0.2 amu) was observed for the trimer (M, ) 5+ ions. With the ESI-MS method it was even possible to demonstrate that hybridization of GCN4-pl and LZ peptides produced small amounts of both dimeric and trimeric heteromers. Furthermore, fluorescence quenching experiments confirmed the formation of coiled coil heteromers in solution, and excluded ESI-MS artefacts. Dimeric complexes of several other DNA- and RNA-binding proteins have been recently identified by ESI-MS (Table 2), including the hydrophobic, lipid-binding lung surfactant protein SPC [22]. These results lend promise to the ES1 method as a tool for detecting even weak (hydrophobic) interactions in nucleotide-binding proteins. However, a significant problem encountered in concentrationand pH-dependent studies is the strong tendency of leucine zippers to adsorb at polar, fused-silica sample delivery capillaries, leading to partial dissociation of complexes [10,19]. Due to this limitation, quantitative information on thermodynamic stabilities on homo- and heteromeric assemblies cannot be obtained at present. The proportions of complex ions could be significantly increased recently by using pre-coated (polyacrylamide) capillaries [21], thus resulting in a rough correlation of molecular ion abundances of dimers and trimers

Table 2 Identification Protem GCN4-p LZ L7 SAF-A

1

of supramolecular

complexes

of polynucleotlde-binding

571

with the stability of the different coiled coils. A further problem is the general, relative reduction of charge states of multiple charged ions for protein complexes, leading to increasing instrumental demands in the detection of highmass (low charge state) complex ions [lo]. 2.3. Identification of double-stranded oligonucleotides

In contrast to the already extensive work on polypeptides, negative-ion ESI-MS of oligo- and polynucleotides has been found significantly more difficult with regard to obtaining a homogeneous, poly-phosphate backbone and composition of counter ions [9]. Hence, the purification problems, e.g. from contaminating alkali salts, still present some limitations to the molecular weight range amenable. Best results have been obtained at present with aqueous poly-ammonium salts which have permitted the detection of homogeneous multiple charged M”- ion series in sequences of up to -80-100 bases [5]. Despite these yet existing limitations, identification of intact double-stranded oligonucleotides has been reported in recent studies [8-lo]. The ES1 spectrum of the duplex 24-mer palindromic recognition sequence of the GCN4 leucine zipper (GCNCU/-L), prepared by hybridization in ammonium acetate, is illustrated in Fig. 3 in comparison to a single-strand sequence. Unequivocal identification of the duplex is obtained (as noted above) by the specific odd-charged ion [M 9H]“-, whereas the masses of the 8-charged duplex ion and the 4-charged monomer ions would not be resolved at the resolution of the quadrupole analyzer employed. However, the charge state distributions in Fig. 3b suggest the nearly quantitative formation of the duplex which was ascertained by independent dissocia-

proteins

by ESI-MS

Mol. wt.

Function/localization

Supramolecular

7165 -3400 12 460 25 100

Transcription activator Leucine zipper peptides Autoantigen Nuclear matrix protein

DNA

binding

RNA DNA

binding binding

association

Complex Dimer Dimer, Dimer Dimer

identified

trimer

M. Przybylski et al. I Toxicology Letters 82183 (1995) 567-575

572

a 5 d(CCG

b AAA AAT GAG TCA TCC GCT GCG) 3

1000

2000

5’ d(CCG AAA AAT GAG TCA TCC GCT GCG) 3 3’ d(GGC TTT TTA CTC AGT AGG CGA CGC) 5

nlh

2000

m/z

Fig. 3. Negative ion ES1 mass spectra of (a) single-strand 24-mer oligonucleotide GCN4-U; (b) double-stranded GCN4-U/-L hybridization product. Solvent, 5 mM NH,OAc:acetonitrile, 91 (pH 6.0).

tion studies and electrophoresis. Furthermore, successful studies of several synthetic oligonucleotides with different sequence/base compositions (see examples in Table 3) showed remarkable stabilities of the duplex forms under ES1 conditions relative to leucine zipper polypeptide complexes. These results suggest the possibility for determining duplex melting temperatures using ESI-MS which has been addressed in first model studies [23]. 2.4. Characterization of nucleotide complexes of EF-TuIEF-Ts

The successful characterization of both supramolecular polypeptide and protein complexes and of double-stranded (and higher-order [7]) oligonucleotides appears to provide, in principle, Table 3 Examples

of double-stranded

Oligonucleotide”

GG, d(T),/d(A), d(T),Jd(A),,, d(20mer) I/II GCN4-L/-U

oligo-deoxynucleotide

hybridization

Mol. wt. (D)h

the basis for directly probing protein-nucleotide interactions in solution under ESI-MS conditions. As noted above, possible effects on charge structures of proteins upon polar interaction with a nucleotide backbone are still unknown, as are the resulting multiple charged ions detectable by ESI-MS. Initial model studies on leucine zipper peptide complexes with complementary oligonucleotides did not yield detectable high chargestate ions within a limited mass range (m/z 2000), indicating the requirement for using suitable high mass analyzers (such as time-of-flight instruments) for detecting complex ions of low charge numbers. However, the suitability of ESI-MS to analyze polar nucleotide complexes in proteins could be demonstrated recently in studies to characterize the nucleotide and protein binding regions in the EF-Tu from T. thermophilus [24]. The transition of EF-Tu from an ‘inactive’ GDP, to the GTP binding form upon interaction with the nucleotide exchange factor EF-Ts was characterized by selective chemical modification and by direct mass spectrometric analysis of the EF-Tu/-Ts and GDP complexes [25]. This study provided the identification of Lys residues for distinct nucleotide binding regions (see Fig. 4). As an example, ES1 spectra of free and GDP-bound forms of EF-Tu are compared in Fig. 4 which revealed a homogeneous ion series of a 1:2-EFTu/GDP complex, under the conditions em-

products

identified m/r

Single strands

Duplex

2411/2411 2371 I2443 298013070 582716410 736017347

4822 4814 6050 12237 14707

1607 1624 1209 2446’ 1638

by negative-ion

ESI-MS

Duplex

ions’

Charge

state”

3 3 5 5 9

dC4G4, self-complementary 5’-dCCCCGGGG-3’: * Oligonucleotide sequences: 3’-GGAAGGAGGGAGAGAGGAGG-5’: GCN4-L/-U, dCC’ITCCTCCCTCTCTCCTCC-3’. TCATTITTCGG-3’. 3’-dGCGTCGCCTACIGAGTAAAAGCC-5’. b Monoisotopic molecular weights without counter-ions. ’ Duplex ions observed as [M - nH]“-. d Most abundant charge states observed within a mass range of approximately 2000. ’ Average molecular weight determined from duplex ions. f Duplex ion obtained with extended mass range quadrupol mass analyzer; see Ref. [9].

Mol. wt.’ 4824 4872 6052 12297 14714

d(20mer-Ii-II), 5’-dCGCAGCGGATGAC-

5’-

M. Przybylskl et al.

SY

A 0

(EF-TU + 2 GDP] [EF-Tu]

-

I Toxicology Letters 82183 (1995) 567-575

nucleotide

MW 45497 MW, 44632

a

effect of different solution structures on multiple charged ions in the gas phase. Further model studies on the chemical preconditions for formation and ESI-MS analysis of polypeptide-nucleotide complexes should lead to rapid improvements and are carried out at present in our laboratory. A key feature will be the investigation of high mass (low charge state) ions of complexes using suitable mass spectrometric instrumentation [26]. Applications to be anticipated include the use of ESI-MS as a tool for studying transcription (or translation) processes, receptor interactions, and the specificity of polynucleotide recognition at a molecular level. Furthermore, the direct analysis by ESI-MS is expected to be a useful complement for the characterization of protein-nucleotide interactions using chemical modification procedures. 4. Experimental

Fig. 4. ES1 mass spectra and structure model of (a) free EF-Tu; (b) EF-Tu complexed with GDP The residue numbers denote lysine residues found shielded in the EF-Tu/ GDP complex towards acetylation (261.

ployed. Likewise, the molecular stoichiometry could be directly determined by ES1 spectra of complexes prepared with di- and trinucleotide co-factors. 3. Conclusions

and perspectives

Successful analyses by ESI-MS have been obtained recently for a variety of non-covalent supramolecular complexes of biomacromolecules and different types of binding, as demonstrated by coiled coil leucine zipper polypeptides and double-stranded oligonucleotides. This unexpected feature of the ES1 method appears to open exciting perspectives for the molecular characterization of biomacromolecular interaction, with the consideration that little is still known about ion formation mechanisms and the

573

procedures

Synthetic leucine zipper peptides employed in this study were prepared on an Applied Biosystems-430A, or a semi-automated ABIMED synthesizer, using Fmoc protection strategy as described previously [21]. Final purification of peptides was achieved by semi-preparative C,-reversed phase HPLC. Oligodeoxynucleotides were synthesized on a Biosearch Cyclone DNA synthesizer using phosphoramidite chemistry and purified by C,,-HPLC before removal of the 5’-protecting group. Hybridization experiments were carried out in 1 M ammonium acetate by annealing at lO”C/h cooling from 80 to 20°C. Final desalting was performed on a Sephadex-G25 column with 10 mM ammonium acetate. ESI-MS was performed with a Vestec-201A quadrupole mass spectrometer (Vestec, Houston, TX) equipped with a ‘thermally-assisted’ electrospray interface [27]. The ion-spray interface temperature was approximately 40°C for all measurements. The mass analyzer with a nominal m/z range of 2000 was operated at unit resolution. An electrospray voltage at the tip of the stainless steel capillary needle of 2-2.2 kV and nozzle-repeller voltage difference of typically lo-50 V were employed. Mass calibration was performed with the 8 + ,

574

M. Przybylski

et al. / Toxicology

9 + and 10 + charged ions of hen egg white lysozyme and raw data analyzed by a Tecnivent vector-2 data system. High resolution ESI-MS measurements were performed with a Jeol JMS102X double focusing sector instrument equipped with an Analytica electrospray interface (Analytica, Branford, MA) at a mass resolution of -7000. Peptide solutions (lo-100 PM) were prepared in 2 mM ammonium acetate:methanol (9:1), and the pH adjusted with acetic acid and aqueous ammonia. Sample delivery was carried out by infusion through a 50-,um fused silica capillary at a flow rate of 4 pllmin using a Harvard-44 infusion pump. Acknowledgements

We thank Dr Alain van Dorsselaer, Louis Pasteur-Universite Strasbourg for helpful discussions on ESI-MS procedures. The assistance of Dr B. Musselmann and Dr J. Tamura, Jeol Inc., Boston, with high-resolution ES1 spectra is gratefully acknowledged. This work was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, the Fond der Chemischen Industrie, and the European Union within the EUnetwork ‘Peptide and Protein Structure Elucidation by Mass Spectrometry’. References [ll Burlingame, A.L. (Ed.) (1990) Biological Mass Spectrometry. Elsevier, Amsterdam. PI Smith, R.D., Loo, J.A., Ogorzalek-Loo, R.R., Busman, M. and Udseth, H.R. (1991) Principles and practice of electrospray-ionization mass spectrometry for large polypeptides and proteins. Mass Spectrom. Rev. 10. 359-401. [31 Karas. M., Bahr, U. and Giepmann, U. (1991) Matrixassisted laser desorption-ionization mass spectrometry. Mass Spectrom. Rev. 10, 335-357. (41 Roepstorff, P., Nielsen, P.F., Klarskov, K. and Hojrup, P. (1988) Applications of plasma desorption mass spectrometry in peptide and protein chemistry. Biomed. Environ. Mass Spectrom. 16, 9-18. [51 Przybylski, M. and Glocker, M.O. (1995) Electrospray mass spectrometry of non-covalent complexes of biomacromolecules - new analytical perspectives for supramolecular chemistry and molecular recognition processes. Angew. Chem. Int. Ed. (in press).

Letters 82183 (1995) 567-575

(61 Ganem. B. and Henion, J.D. (1993) Detecting noncovalent complexes of biological macromolecules: new applications of ion-spray mass spectrometry. Chemtracts 6, l-22. [71 Smith, R.D. and Light-Wahl, K.D. (1993) The observation of non-covalent interactions in solution by electrospray ionization mass spectrometry: promise, pitfalls and prognosis. Biol. Mass Spectrom. 22, 493-501. [81 Ganem, B., Li, Y.T. and Henion. J.D. (1993) Detection of ohgonucleotide duplex forms by ionspray mass spectrometry. Tetrahedron Lett. 34, 1445-1448. 191 Light-Wahl, K.J., Springer, D.L., Winger, B.E.. Edmonds. C.G., Camp, D.G., Thrall, B.D. and Smith, R.D. (1993) Observation of a small oligonucleotide duplex by electrospray ionization mass spectrometry. J. Am. Chem. Sot. 226, 803-804. 1101 Przybylski, M. (1995) Mass spectrometric approaches to the characterization of tertiary and supramolecular structures of biomacromolecules. Adv. Mass Spectrom. 13, (in press). [ill Baca. M. and Kent, S.B.H. (1992) Direct observation of a ternary complex between the dimeric enzyme HIV-l protease and a substrate-based inhibitor. J. Am. Chem. sot. 114. 39x-3993. 1121 Leize, E., van Dorsselaer. A., Kramer, R. and Lehn, J.-M. (1993) Electrospray mass spectrometry of the selfassembly of a capped polymetallic complex. J. Chem. Sot. Chem. Commun. 990-993. 1131 Kebarle. P. and Tang, L. (1993) From ions in solution to ions in the gas phase. Anal. Chem. 65, 972A-985A. [I41 Glocker, M.O.. Borchers, C., Fiedler, W., Suckau. D. and Przybylski, M. (1994) Molecular characterization of the surface topology in protein tertiary structures by aminoacylation and mass spectrometric peptide mapping. Bioconj. Chem. 5, 583-590. [I51 Miranker, A., Robinson, C.V., Radford, SE., Aplin. R.E. and Dobson. C.M. (1993) Detection of transient protem folding populations by mass spectrometry. Science 262. 8966900. [I61 Hurst, R.S. (1994) Transcription factors 1:bzip proteins. In: P. Sheterhne (Ed.), Protein Profile, Academic Press, London. pp. 123-168. [I71 Ellenberger. T.E., Brandl, C.J., Struhl, K. and Harrison, S.C. (1992) The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell 71, 12231237. [I81 O’Shea, E.K., Klemm. J.D., Kim, P.S. and Alber, T. (1991) X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254, 539-544. [I91 Li. Y.-T., Hsieh. Y.-L., Henion, J.D., Senko, M.W., McLafferty, F.W. and Ganem, B. (1993) Mass spectrometric studies on noncovalent dimers of leucine zipper peptides. J. Am. Chem. Sot. 115, 840998413. 1201 Lovejoy, B.. Choe, S., Cascio, D., McRorie, D.K.. DeGrado, W.F. and Eisenberg, D. (1993) Crystal structure of a synthetic a-helical bundle. Science 259, 12881293.

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[21] Wendt, H., Diirr, E., Thomas, R.M., Przybylski, M. and

Bosshard, H.R. (1995) Characterization of leucine zipper complexes by electrospray mass ionization spectrometry. Protein Sci. (in press). [22] Przybylskt, M.. Glocker, M.O., Maier, C., Borchers, C., Ddrr. E., Fiedler, W., Kast, J., Wendt, H. and Bosshard, H.R. (1994) Direct characterization of supramolecular complexes of polypeptides and proteins by electrospray mass spectrometry. In: H.L.S. Maia (Ed.), Peptides 1994. Escom, Leiden, pp. 42-43. [23] Goodlett, D.R., Hardin, C.C., Corregan, M. and Smith, R.D. (1993) Estimation of quadruplex DNAT, by electrospray-ionization-mass spectrometry. Proc. 41st Conf. Am. Sot. Mass Spectrom. 258. [24] Berchtold. H., Reshetnikova, L., Reiser, C.A.O.,

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Schirmer, N.K., Sprinzl, M. and Hilgenfeld. R. (1993) Crystal structure of active elongation factor Tu reveals major domain rearrangements. Nature 365, 126-132. [25] Glocker, M.O., Neck, S.. Sprinzl, M. and Przybylski, M. (1995) Mass spectrometric characterization of non-covalent T’T EF-TulTS complexes. Proc. 43rd Conf. Am. Sot. Mass Spectrom. (in press). [26] Finch, J.B., Cody, R.B., Tamura, J. and Musselman, B.D. (1994) Electrospray and LC/MS on magnet sector mass spectrometers: improvements in performance. Proc. 42nd Conf. Mass Spectrom. Allied Top. 753. [27] Allen, M.H. and Vestal, M.L. (1992) Design and performance of a novel electrospray interface. J. Am. Sot. Mass Spectrom. 3, 18-26.