J. Pepride R e s . 5 1 , 1 9 9 8 , 4 4 4 4 5 1 Prinred in rhe United States ofAmerica - ail rrghrs reserved

CoDvriaht 0 Munksaaard. 1998 1 , Y

JOURNAL OF PEPTIDE RESEARCH ISSN 1397-002X

An .alysis of synthetic peptides by capillary zone electrophoresis in organic/aqueous buffers

CHARLEEN MILLER and JEAN RIVIER

Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California, USA

Received 21 October, revised 18 December 1997, accepted for publication 17 January 1998

Whereas synthetic peptides have been routinely analyzed for purity by reverse phase high performance liquid chromatography (RPHPLC) for a number of years, it is only in the last decade that the use of capillary zone electrophoresis (CZE) in aqueous buffers has been taken advantage of as an orthogonal method for the detection of impurities. However, we have found that hydrophobic amino acids and peptides often migrate as very broad, tailing absorbances or even precipitate in the aqueous buffers during CZE analysis. As a result, alternative buffer systems containing organic modifiers were sought. Varying concentrations of acetonitrile, methanol and isopropanol in sodium phosphate and triethylammonium phosphate buffers were used to study their effects on the electrophoretic migration of several synthetic peptides [gonadotropin releasing hormone (GnRH), corticotropin releasing factor (CRF) and analogs] and an enantiomeric synthetic amino acid. The organic/aqueous buffers used to obtain the best conditions for separation of porcine gonadotropin-releasing hormone (GnRH) and chicken I1 GnRH were then used to optimize a separation of nine native forms of GnRH decapeptides. Interestingly, several of these GnRHs have identical formal charges and yet could be separated. This suggests a mixed mechanism of separation that discriminates not only on the basis of peptide charge and structure but also of adsorptive properties (Van der Waals forces, dipole-dipole interactions and hydrogen bonding) of the capillaries. 0 Munksgaard 1998.

Key words: capillary zone electrophoresis; mixed organic/aqueous buffers; peptides; GnRH; CRF, HPLC

During routine characterization of synthetic peptides us- ing capillary zone electrophoresis, (CZE) impurities were uncovered that had not been detected using RPHPLC under a number of optimized conditions thus validating the need for multiple analytical techniques and solvent systems for the characterization of peptides (1-5). A re- view of the literature shows that CZE analysis of pep- tides is most often accomplished using aqueous buffers based on mineral salts at low pH (6-8). Because of our interest in uncovering and quantitating difficult-to-detect impurities, we have extended the range of buffers to in- clude organic salts such as the triethylammonium phos- phate buffer (TEAP) at different pHs, buffers which we have found to be particularly useful for the separation of peptides both analytically and preparatively using re- verse phase (9) as well as gel exclusion chromatogra- phies (10). In fact, we have found that the resolving power of CZE is increased in the presence of TEAP buffer ( 5 ) although for certain classes of peptides severe peak broadening is apparent. We had also found that the addition of acetonitrile into the buffer systems used for ion exchange chromatography as well as size exclusion

chromatography could be extremely beneficial (2, 11, 12). We hypothesized that acetonitrile increases the solu- bility of the peptides and facilitates partitioning among phases (stationary and mobile). In CZE, the peptides must remain soluble and their interactions with the cap- illary wall should be minimized (see ref. (1 3 ) for review of all parameters influencing separations in CZE). Ac- cordingly, we have used capillaries coated with a neu- tral hydrophilic coating and investigated the effects of including varying amounts of acetonitrile in sodium phosphate and TEAP at acidic pH. Additionally we have investigated the use of methanol and 2-propanol as co- solvents. Buffer modifiers have been used extensively and can be divided into three groups: inorganic, organic and cyclodextrins. An exhaustive review of the literature by Issaq et al. (13) refers to works by a number of in- vestigators whereby organic co-solvents will improve analyte solubility, detector sensitivity, separation, reso- lution, and help control electrososmotic flow mostly through increases or decreases of viscosity of the me- dium and decrease analyte-wall interactions. Addition- ally, organic modifiers will reduce the dielectric constant

444

CZE in organic/aqueous buffers

TABLE 1 Structures of peptides"

and the zeta potential (13). A recent report by Zhu et al. (1 4) illustrates the use of 50% acetonitrile-containing buffer for such use as solubilizing hydrophobic relatively small peptides. From the illustration of their results, it was clear that the addition of acetonitrile had no signifi-

fluencing Selectivity and recovery. In fact, shortened

Mammalian GnRH pGlu-His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-

Chicken GnRH I pGlu~His~Trp-Ser-Tyr-Gly-Leu-Gln-Pro-

Sea Bream GnRH pG]u-His-Trp-Ser-Tyr-G]y-Leu-Ser-Pro-

GIY-NH~

cant advantage in terms of enhancing resolution and in- Gly-NH2

retention times (most likely due to lowered viscosity of GIv-NH, the buffer) resulted in poorer separation than had been found in the absence of acetonitrile. The main goals of this study were two-fold: (a) identify highly resolving and reproducible CZE conditions for small and large, hy- drophobic and hydrophilic peptides and (b) more particu- larly, find out whether one could use CZE for the separation of all known native GnRHs; conditions that would be more sensitive than the ones found using HPLC (4). Whereas the first goal was purely of analyti- cal interest, the second was meant to develop an analyti- cal tool with increased sensitivity that would allow us to identify novel GnRH molecules in a minimum amount of tissue extracts.

RESULTS AND DISCUSSION

The decapeptides gonadotropin-releasing hormones (GnRHs) (Table 1) play a major role in the modulation of reproductive functions. The GnRH family is neither related to other peptide families nor a member of a pep- tide superfamily. However, the GnRH family is ever growing with nine members isolated from various ver- tebrates (15). One or more of the nine GnRH forms is found in individual species from all classes of verte- brates. All of these peptides have conserved residues at positions 1, 2, 4, 9 and 10. Because more than one GnRH is found in most species (with the exception of mammals) the role of such molecules is the source of much interest for the understanding of the evolution of the reproductive system. Studies of species with mul- tiple forms of GnRH in the brain have shown that each endogenous form of GnRH can release gonadotropins in vitro, provided the pituitaries are from the same spe- cies (16, 17). However, the multiple GnRH forms in the brain of a single species are assumed to have func- tions in addition to that of releasing pituitary hormones based on variation in their structures [see Sherwood et al. (18)] and brain locations [see Davis et al. (19)]. Little is known about these functions (18). Addition- ally, GnRH superagonists are now available for thera- peutic use, while the parent competitive antagonists still suffer in terms of their potential clinical use from rela- tively low potency and the fact that some of them release histamine in a variety of tests (20) includin in human (21). The Nal-Glu antagonist ([Ac-DNal , ~Cpa~,~Pa1~,Arg~,4-(p-methoxybenzoyl)-~-2-Abu~, ~Ala"] GnRH) (22) studied in this paper is an antago- nist that was extensively tested in humans (23).

Corticotropin releasing factor (CRF) is a 41-resi-

8

- - Salmon GnRH pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-

Dogfish GnRH I11 pGlu-His-Trp-Ser-His-Gly-Trp-Leu-Pro

Catfish GnRH I pGlu-His-Trp-Ser-His-Gly-Leu- Asn-Pro

Chicken GnRH I1 pGlu-His-Trp-Ser-His-Gl y-Trp-Tyr-Pro

Lamprey GnRH I pGlu-His-Tyr-Ser-Leu-Glu-Trp-Lys-Pro

Lamprey GnRH 111 pGlu-His-Trp-Ser-His-Asp-Trp-Lys-Pro

Nal-Glu Antagonistb Ac-D2Nal-D4Cpa-D3Pal-Ser-Arg-~Xaa- Leu-Arg-Pro DAla-NH2

rCRF Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu- Asp-Leu- Thr-Phe-His-Leu-Leu- Arg-Glu-Val-Leu- Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala- Gln-Gln- Ala-His-Ser- Asn- Arg-Lys-Leu- Met-Glu-Ile-Ile-NH2

G 1 y - N H 2

Gly-NHz

Gly-NH2

G 1 y - N H 2

Gly-NH2

Gly-NH2

aDifferences from the structure of mammalian GnRH are shown in bold characters. boXaa = o-2-amino-5-oxo-5-(2 or 4-methoxyphenyl)pentanoic acid.

due peptide amide (24) (see Table 1) which stimu- lates the release of ACTH in vitro and in vivo (24, 25) and acts within the brain to modulate a wide range of stress responses (25) as demonstrated by the extensive use of competitive antagonists such as the a-helical CRFd9-41) (26) or analogs arising from the [ ~ P h e ' * , N l e ~ ~ , ~ ]hCRF(1241) lead (27). CRF and ana- logs are good representatives of a class of peptides which we consider large in size and hydrophobic giv- ing poor capillary electrophoretic resolution even when using acidic buffers and coated capillaries. Be- cause biological results are often highly dependent on the purity of the peptides used, we are regularly upgrading our analytical tools for the identification of closely related impurities.

Current academic and pharmaceutical research has fo- cused on the characterization of natural extracts (plants, marine organisms or bacterial broths) and on the devel- opment of methodologies for generating chemical diver- sity (peptide or peptidomimetic libraries) for the discovery of new bioactive leads. The latter approach depends on automation of chemical methods for solid-phase synthe- ses and the identification of novel scaffolds. Among them, P-methyl amino acids (28) are of unique interest for the design of rigid peptide-based analogs in that they have built-in rotational constraints of their side chains which will complement constraints brought about by the intro-

445

C. Miller and J. Rivier

duction of cycles. Such rigid analogs are particularly im- portant for the definition of the bioactive conformation of peptide hormones. We were particularly interested in these amino acids and the effect of their introduction in GnRH antagonists and somatostatin analogs. Unlike Dharanipra- gada et al. (29) who obtained optically pure P-methyl- phenylalanine (P-Me-Phe) using asymmetric synthesis, we opted for a combination of crystallization steps for the separation of the diastereomers and enzymatic resolution or fractional crystallization of optically active salts for the separation of the enantiomers. During these studies we used CZE as a means to follow progress in our resolu- tion efforts (Simon, L. and Rivier, J., in preparation).

Five synthetic peptides (Nal-Glu antagonist, two closely related native GnRHs, and two members of the CRF family) and an enantiomeric synthetic amino acid (P-Me-Phe) were first selected for the difficulty we had found to separate them using CZE (GnRHs) or for their unique (poor) electrophoretic properties (CRF and ana- logs). Figure l illustrates a separation of such molecules under standard conditions.

The discovery, using CZE, of a significant amount of a contaminant in the clinical preparation of the Nal- Glu antagonist was certainly unexpected and later un- derstood, as the contaminant is a positional isomer ([Ac-DNal',DC aZ,~Pa13,ArgS,4-(o-methoxybenzoy~)-~- 2-Abu6,~Ala' 3 GnRH). Although the ortho isomer could be observed using standard conditions (Fig. l), it is only with the use of the TEAP buffer that we could

B

10

$ f 8 5 2 N

2

0

-

k f z t

.- D

I - - (? -

-- d 30 35 40

15 20 25 Time (rnin)

FIGURE 1 CZE trace of test mixture using aqueous sodium phosphate. Supelco CElect-PI75 Capillary; (50 cm x 75 pm) at 30°C; Voltage: 12 kV; Current: 64 FA; Buffer: sodium phosphate; 100 mM, pH 2.5; AUFS 0.01 at 214 nm.

get good resolution (almost baseline separation) and therefore good quantitation in a reliable way (Fig. 2A). Because both isomers have the same formal charges, one may wonder what is the mechanistic basis for their separation. Whatever it is, it is likely to be the same mechanism that leads to the separation of the two enan- tiomers of P-Me-Phe (Fig. 1).

6

E C * (v

v) LL 3

E

Y

$ 3

a

0

A

20

6

E C d nl

v) LL 3

Y

z 3

a E

0

FIGURE 2

25 30 35 Time (min)

u, 5 B

I l b 15 20

Time (min)

CZE trace of test mixture using TEAP. (A) Supelco CElect-Pi75 Capillary; (50 cm x 75 pm) at 30°C; Voltage 20 kV; buffer: TEAP; 100 mM, pH 2.5; AUFS 0.006 at 214 nm. (B) same as above; Buffer: TEAP; 100 mM, pH 2.5 in (85: 15, H*O:CH3CN).

446

Illustrated in Figure 1 is the broadening of bands ob- served for CRF-like molecules which is further aggra- vated by the use of the TEAP buffer (Fig. 2A) and nicely solved with the addition of 15% CH3CN (Fig. 2B). Whereas the use of TEAP tends to increase mi- gration time as compared to the use of a sodium phos- phate-based buffer (Fig. l ) , addition of CH3CN (lowering the viscosity of the buffer) reduces migration in the capillary by more than 30%, a saving in time that may have commercial advantages. Significant sharpen- ing of the CRF bands is also observed if CH3CN is added to the buffer shown in Figure 1, however no in- crease in resolution between the two Nal-Glu isomers or between the two CRFs is observed (data not shown) and migration times are shortened as expected. We pro- pose that the addition of CH3CN in the running buffer increases solubility of the peptide and modifies by an as yet not identified mechanism the physico-chemical properties of the capillaries' walls/coatings (see Issaq et ai. (13) for additional characterization of capillary coatings). We found that the graded addition of isopro- pan01 to sodium phosphate or TEAP buffer causes an increase in migration time; the opposite of what is seen with methanol and acetonitrile. This may be due to the increased viscosity of the isopropanol-containing buffer. Although the shortest migration times were achieved with smaller percentages of isopropanol, the peak shapes of CRFs were especially broad. Another interesting observation was that the CRFs co-migrated at 25% isopropanol in sodium phosphate and separated, reversing migration order as the concentration of iso- propanol was increased (data not shown). Because of these effects, the use of isopropanol as a co-solvent for CZE is not recommended.

In Figure 1 we also show the potential of CZE for the quantitative determination of diastereomeric ratios for /3-Me-Phe and other similarly hydrophobic amino acids. Migration times could be reduced to ca. 30 and 20 min. by the addition of 15% CH3CN in the buffer or by increasing voltage to 20 kV [data not shown and Miller and Rivier respectively (4)]. The diastereomers were included in the study as the presence of organic modifiers in the buffer was thought to influence their separation much differently than that of the peptides. This was not found to be the case.

As mentioned earlier, we could find HPLC condi- tions under which all nine known native GnRH (see Table 1 for sequences) could be separated (Fig. 3). As can be seen, the conditions include the use of a nar- row-bore column, TEAP pH 6.5/acetonitrile and CI8 support at 40°C. The peptides differed by one or sev- eral residues and are mostly baseline resolved. Two of the peptide absorbances were fused but different reten- tion times were identifiable.

We were interested in matching these results using CZE because of possible increase in resolution, increase in sensitivity and shortened time for analysis. As shown

8C

E f

2 Z 4 C w 3

E

0

CZE in organic/aqueous buffers - -

Time (min.)

FIGURE 3 Separation of synthetic GnRHs from different species. Load: 0.2 pg each GnRH; Vydac CIS (5 mm particle size) column (0.21 x 15 cm). Buffer was TEAP pH 6.5; flow rate, 0.2 mL/min.

in Figure 4, standard conditions showed that the nine peptides migrated in three groups as expected on the ba- sis of the number of basic residues in their composition.

' 25 30 35 40

Time (min)

FIGURE 4 CZE separation of nine synthetic GnRH. Supelco CElect-PI75 Cap- illary; (50 cm x 75 pm) at 30°C; Voltage: 10 kV; Current: 52 pA; Buffer: sodium phosphate; 100 mM, pH 2.5; AUFS 0.01 at 214 nm.

447

C. Miller and J. Rivier

Lamprey 111 GnRH migrated first (three basic residues) followed by a group comprising catfish I, porcine, dog- fish 111, chicken IT and lamprey I GnRHs (all with 2 ba- sic residues in their composition) and the group comprising sea bream, salmon and chicken I GnRHs (all with one basic residue in their composition).

We first sought conditions under which the first two groups of GnRH could be separated. This separation is shown in Figure 5 and takes advantage of the unique properties of the TEAP buffer and methanol. The third group of GnRHs could also be separated using the TEAP buffer and acetonitrile (Fig. 6). TEAP buffer without the addition of organic co-solvents (such as methanol and acetonitrile) results in lengthy migration times. Including 25% methanol was not strong enough organically for separation of the third group so aceto- nitrile was added to TEAP to accomplish the separa- tion within 35 min.

Recently D’Eramo et al. (30) described a chro- matographic system for the separation of five isoforms of GnRH using an ion-exchange HPLC col- umn and isocratic elution; these were porcine GnRH, salmon GnRH, chicken I GnRH, chicken I1 GnRH and lamprey I GnRH. The mobile phase used con-

O L

20 25 30 35 40 Time (min)

FIGURE 5 CZE separation of six synthetic GnRH using TEAP + MeOH. Supelco CElect-P175 Capillary; (50 cm x 75 pm) at 30°C; Volt- age: 25 k V Current: 68 pA; Buffer: TEAP; 100 mM, pH 2.5 in (75:25, H,O:MeOH); AUFS 0.01 at 214 nm.

sisted of sodium phosphate at pH 2.90, acetonitrile and methanol (70:15:15, v/v). There are clear simi- larities in our and D’Eramo’s et al. approach al- though we can surmise that a CZE-based approach will be more sensitive than one based on ion ex- change chromatography.

The most dramatic results derived from the use of an organic co-solvent however were found during the separation of CRF and analogs. As shown in two ex- amples (Figs. 7 and 8), going from 15% acetonitrile to 25% acetonitrile in the composition of the buffer has dramatic consequences in terms of both resolu- tion and recovery of the peptides. We have found that routine addition of 15% acetonitrile to our buffers is valuable in increasing resolution and decreasing run times for analysis of most synthetic peptides. The ac- etonitrile concentration may be increased if band broadening is still apparent or the concentration may be reduced in the case of highly basic peptides.

5

2.5

- S Q, Y V .- f

25 30 35 Time (min)

FIGURE 6 CZE separation of synthetic GnRH using TEAP + CH3CN. Supelco CElect-P175 Capillary; (50 cm x 75 pm) at 30°C; Voltage: 25 kV; Current: 76 pA; Buffer: TEAP; 100 mM, pH 2.5 in (75:25, H,O:CH,CN); AUFS 0.005 at 214 nm.

448

3.0

E c A

20 25 30 Time (min)

4.

E c w cv

a lL 3

E

r

is 2.1

a

B

20 25 30 Time (min)

FIGURE 7 CZE trace of a CRF antagonist in the presence of 15% and 25% CH3CN. (A) Supelco CElect-P175 Capillary; (50 cm x 75 pm) at 30°C; Voltage: 13 kV; Buffer: sodium phosphate; 100 mM, pH 2.5 in (85:15, H,O:CH,CN); AUFS 0.003 at 214 nm. (B) same as above; Voltage: 13 kV, Buffer: sodium phosphate; 100 mM, pH 2.5 in (75:25, H,O:CH3CN); AUFS 0.004 at 214 nm.

CZE in organic/aqueous buffers

A

20 25 30 Time (min)

B

~

20 25 30 Time (min)

FIGURE 8 CZE trace of an &-helical CRF antagonist in the presence of 15% and 25% CH3CN. (A) Supelco CElect-P175 Capillary; (50 cm x 75 ym) at 30°C; Voltage: 15 kV, Buffer: sodium phosphate; 100 mM, pH 2.5 in (85:15, HzO:CH3CN); AUFS 0.003 at 214 nm. (B) same as above; Voltage: 15 kV, Buffer: sodium phosphate; 100 mM, pH 2.5 in (75:25, H20:CH3CN); AUFS 0.01 at 214 nm.

449

C. Miller and J. Rivier

CONCLUSION

TEAP was investigated as an electrophoretic buffer in addition to our standard sodium phosphate. Its usefulness in the separation of NPY diastereomers has been documented (5) but the use of TEAP in combination with organic modifiers had not been explored. No single organic modifier + aqueous buffer used in these experiments was found optimal for the separation of all nine forms of GnRH. Aque- ous sodium phosphate was unable to resolve two pairs of GnRHs: catfish GnRH I and porcine GnRH; chicken GnRH I and salmon GnRH; while aqueous TEAP delayed the migration so much that none of the peptides were detected during a 50 min run at a voltage of 20 kV. Only when utilizing TEAP in the presence of 25% organic modifier were separations successful. As shown in Figure 5 , a concentration of 25% methanol in TEAP pH 2.5 was found optimal for the separation of porcine GnRH, catfish GnRH I, dog fish GnRH 111, lamprey GnRH I and chicken GnRH 11. TEAP in the presence of 25% acetonitrile with an applied voltage of 25 kV provided the ap- propriate conditions to obtain separation of salmon GnRH, sea bream GnRH and chicken GnRH I in 33 min as shown in Figure 6.

EXPERIMENTAL

Apparatus The instrumentation used has been described previously (10, 3 1). The capillary electrophoresis system consisted of the P/ACE System 2050 (Beckman, Palo Alto, CA) with Chromjet integrator (Spectra-Physics, Indianapo- lis, IN). Spectra were detected at 214 nm. Mass spec- tra were measured on a JMS-HX110 mass spectrometer using liquid secondary ionization (Jeol, Peabody, MA).

Capillaries CElect-P175 capillaries were (50 cm x 75 mm i.d.) from Supelco. The temperature was maintained at 30°C.

Chemicals and reagents All reagents and solvents were of analytical grade (Aldrich Chemical Co, Milwaukee, WI; Fisher Scien- tific, Pittsburgh, PA) and were used without further pu- rification except TFA (Halocarbon, North Augusta, SC) and TEA (Aldrich), which were reagent grade and used without further purification for syntheses and were dis- tilled to constant boiling point for use in chromato- graphic buffers. Phosphoric acid (Fisher Scientific), methanol (Baxter Columbia, MD), isopropanol and ac- etonitrile (Mallinckrodt, Phillipsburg, NJ) were of HPLC grade. Water was obtained from a Milli-Q 4- bowl purification system (Millipore) using in-house distilled water feed.

Buffers The aqueous buffer was 100 mM phosphoric acid ad- justed to pH 2.5 by addition of 2 M sodium hydroxide or 100 mM phosphoric acid adjusted to pH 2.5 by addi- tion of triethylamine. Organic/aqueous buffer was com- prised of 100 mM phosphoric acid in organic/water (v/ v) and adjusted to pH 2.5 by addition of 2 M sodium hy- droxide or triethylamine. Buffers used in the chromato- graphic and electrophoretic systems were degassed and filtered through a 0.22-pm filter Millipore filter.

Peptide synthesis All peptides were assembled using standard solid phase peptide synthesis (SPPS) techniques on p-rnethylbenz- hydrylamine (MBHA) resin, using previously described protocols (32, 33 j . Briefly, tert-butyloxycarbonyl (BOC) was used for N-terminal protection and deblocked with trifluoroacetic acid (TFA) in the pres- ence of ethanedithiol (EDT). Most couplings employed 1,3-diisopropylcarbodimide (DIC j as primary coupling reagent; more difficult cycles required the use of benzotriazol- 1 - yl-ox ytris (dimethy1amino)phosphonium hexafluorophosphate (BOP) in N-methylpyrrolidinone or DMF in the presence of excess of diisopropyl- ethylamine (DIPEA). Coupling times were generally 1 h, monitored by qualitative Kaiser test, repeating cou- pling steps as necessary. After TFA deprotection, resin washes included 2-propanol (containing 1 % EDT), tri- ethylamine (10% in DCM) for neutralization of pep- tide, MeOH and DCM. The protected peptide was cleaved from the resin by anhydrous HF in the pres- ence of 3% anisole at 0°C for 90 min. Volatiles were removed in vacuuo. The crude peptides were precipi- tated with anhydrous ethyl ether and filtered to remove ether soluble non-peptide materials, and extracted in water. After lyophilization, crude peptides were puri- fied by preparative reverse-phase HPLC (34) on a Wa- ters DeltaPrep LC 3000 system equipped with a Waters 1000 Prep Pak Module and a Shimadzu SPD-6A vari- able wavelength UV detector. The cartridges used were hand packed, in-house, with Waters polyethylene sleeves and frits and Vydac CI8 packing material (15- 20 pm particle size, 30 nm pore size). The material was eluted first using a linear TEAP/60% MeCN (pH 2.25 or 5 . 2 ) buffer system gradient; acceptable fractions were pooled, reloaded onto the preparative cartridge and desalted in 0.1 % TFA. Final products were >95% pure by HPLC analysis.

Peptide characterization Purified peptides were subjected to amino acid analy- sis [hydrolysis in 4 N methanesulfonic acid at 110°C for 24 h, followed by ion exchange chromatography and post column derivativization with o-phthalalde- hyde], and liquid secondary ionization-MS analysis measured with a Jeol JMS-HX110 double focusing mass spectrometer (Jeol, Tokyo, Japan) fitted with a Cs'

450

gun. Samples were added directly to a glycerol and 3-nitrobenzyl alcohol (1: 1) matrix.

Peptide preparation Peptides were dissolved in 0.1% TFA containing 10% acetonitrile and solutions stored frozen. The nine GnRH decapeptides were introduced by three injections of so- lutions containing the following: lamprey GnRH 111 (0.04 mg/mL) and catfish GnRH I (0.02 mg/mL); por- cine GnRH (0.017 mg/mL), dogfish GnRH 111 (0.033 mg/mL), chicken GnRH I1 (0.033 mgfmL) and lamprey GnRH I; sea bream GnRH (0.022 mg/mL), salmon GnRH (0.033) and chicken GnRH I (0.044 mg/mL). Injections were for 2 sec, 3 sec and 1.5 sec respectively.

ACKNOWLEDGMENTS We thank the following for synthesis and purification of the pep- tides: Ron Kaiser, Laura Cervini, Sabine Lahrichi, Robert Galyean, John Porter, Michel Ibea and Guangcheng Jiang. We thank Debbie Johns for manuscript preparation. This work was supported by NIH grant HD13527, DK26741 and the Hearst Foundation.

REFERENCES 1 .

2.

3.

4. 5.

6.

7.

8.

9. 10. I I .

12. 13.

14.

15.

16.

Miller, C. L., Hernandez, J.-F., Dykert, J. and Rivier, J. E. (1990) in ANABIOTEC '90, Burlingame/San Francisco, CA,

Miller, C., Hernandez, J.-F., Craig, A. G., Dykert, J. and Rivier, J. (1991) Anal. Chimica Acta 249,215-225 Hoeger, C., Theobald, P., Porter, J., Miller, C., Kirby, D. and Rivier, J. (1991) in Methods in Neurosciences (Conn, P. M., ed.), vol. 6, pp. 3-27, Academic Press, Orlando, FL Miller, C. and Rivier, J. (1996) Biopolymers 40,265-317 Kirby, D. A., Miller, C. L. and Rivier, J. E. (1993) J . Chromatog. 648,257-265 Caprioli, R. M., Moore, W. T., Martin, M. and DaGue, B. B. (1989) J . Chromatog. 480,247-257 Firestone, M. A., Michaud, J.-P., Carter, R. H. and Thormann, W. (1987) J . Chromatog. 407,363-368 Grossman, P. D., Colburn, J. C. and Lauer, H. H. (1989) Analyt. Chem. 61, 11861194 Rivier, J. (1978) J . Liq. Chromatog. 1, 343-367 Rivier, J. E. (1980) J . Chromatog. 202,221-222 Miranda, M. T. M., Craig, A. G., Miller, C., Liddle, R. A. and Rivier, J. (1993) J . Prof. Chem. 12,533-544 Rivier, J. and McClintock, R. (1983)J. Chromatog. 268, 112-119 Issaq, H. J., Janini, G. M. and Chan, K. C. (1995) in Advances in Chromatography (Brown, P. R., Grushka, E., eds.), vol. 35, pp. 101-169, Marcel Dekker, New York, NY Zhu, M., Hansen, D. L., Burd, S. and Gannon, F. (1989) J . Chromatog. 480,311-319 Powell, J. F. F., Zohar, Y., Elizur, A., Park, M., Fischer, W.H., Craig, A.G., Rivier, J.E., Lovejoy, D.A. and Shenvood, N.M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12081-12085 Habibi, H. R., Peter, R. E., Nahorniak, C. S . , Milton, R. C. d. L. and Millar, R. P. (1992) Regul. Pept. 37,271-284

pp. PO4-PO10

CZE in organic/aqueous buffers

17. Schulz, R. W., Bosma, P. T., Zandbergen, M. A,, Van der Sanden, M.C., Van Dijk, W., Peute, J., Bogerd, J. and Goos, H.J. (1993) Endocrinology 133, 1569-1577

18. Sherwood, N. M., Lovejoy, D. A. and Coe, I. R. (1993) Endocrinol. Rev. 14,241-254

19. Davis, M. R. and Fernald, R. D. (1990) J . Neurobiol. 21,

20. Schmidt, F., Sundaram, K., Thau, R. B. and Bardin, C. W. (1984) Contraception 29, 283-289

21. Hall, J. E., Brodie, T. D., Badger, T. M., Rivier, J., Vale, W., Conn, P.M., Schoenfeld, D. and Crowley, W.F., Jr. (1988) J . Clin. Endocrinol. Metab. 67, 524-53 1

22. Rivier, J., Porter, J., Rivier, C., Pemn, M., Comgan, A., Hook, W.A., Siraganian, R.P. and Vale, W.W. (1986) J . Med. Chem. 29, 18461851

23. Urban, R. J., Pavlou, S. N., Rivier, J. E., Vale, W. W., Dufau, M. L. and Veldhuis, J. D. (1990) Am. J . Obstet. Gynecol. 162, 1255-1 260

24. Vale, W., Spiess, J., Rivier, C. and Rivier, J. (1981) Science 213, 1394-1397

25. TachC, Y. and Rivier, C., eds. (1993) Corticotropin-Releasing Factor and Cytokines: Role in the Stress Response, p. 296, The New York Academy of Sciences, New York

26. Rivier, J., Rivier, C. and Vale, W. (1984) Science 224,

27. Hernandez, J.-F., Kornreich, W., Rivier, C., Miranda A,, Yamamoto, G., Andrews, J., Tache, Y., Vale, W. and Rivier, J. (1993) 1. Med. Chem. 36,2860-2867

28. Kazmierski, W. M., Urbanczyk-Lipkowska, 2. and Hruby, V. J. (1994)J. Org. Chem. 59, 1789-1795

29. Dharanipragada, R., Nicolas, E., Toth, G. and Hruby, V. J. (1989) Tetrahed. Lett. 30, 68416844

30. D'Eramo, J. L., Somoza, G. M., Stefano, A., Canosa, L. F. and Fridman, 0. (1996) J . Chromatog. 682,219-224

31. Hoeger, C., Galyean, R., Boublik, J., McClintock, R. and Rivier, J. ( 1 987) Biochromatography 2, 134- 142

32. Barany, G. and Merrifield, R. B. (1980) in The Peptides, Analy- sis, Synthesis, Biology (Gross, E., Meienhofer, J., eds.), vol. 2, pp. 1-284, Academic Press, New York

33. Stewart, J. M. and Young, J. D. (1984) in Solid Phase Peptide Synthesis, 2nd edition, pp. 176, Pierce Chemical Company, Rockford, Illinois

34. Rivier, J., McClintock, R., Galyean, R. and Anderson, H. (1984) J . Chromatog. 288,303-328

1 1 80-1 1 88

889-891

Address:

Jean Rivier, Ph.D. The Salk Institute The Clayton Foundation Laboratories for Peptide Biology 10010 N. Torrey Pines Road La Jolla, California 92037 USA Phone: (619) 453-4100 ext. 1350 Fax: (619) 552-1546 E-mail: Jrivie@salk.edu

45 1