Analytica Chimica Acta, 249 (1991) 215-225

Elsevier Science Publishers B.V., Amsterdam

215

Synthesis, purification and characterization of rat histone H2A ( l-53) -NH,

Charleen Miller, Jean-Francois Hernandez, A. Grey Craig, John Dykert and Jean Rivier *

Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037 (USA)

(Received 8th November 1990)

Abstract

Synthetic peptides (up to 40 residues in length) are routinely purified and characterized by reversed-phase liquid

chromatography (RP-LC). However, as the peptide size is further increased, this technique becomes less efficient in

separating micro-heterogeneities despite the extensive use of columns or buffer systems known to exhibit different

selectivities. As a result, alternative analytical and preparative methodologies were sought for the purification and

characterization of synthetic histone H2A (l-53)-NH,. This 53-peptide was synthesized on a 2,4-dimethoxy-

benzhydrylamine resin using the fluorenylmethyloxycarbonyl strategy. Interestingly, the crude histone, after trifluoro-

acetic acid cleavage from the resin and deprotection, appeared to be remarkably pure. This result being unexpected,

this sample was also analyzed by cation-exchange chromatography using a recently developed narrow-bore Pharmacia

Mono S Precision Column 1.6/5 and an organic modifier (acetonitrile) in the buffer. Under the experimental

conditions that were used, the presence of the desired peptide was determined by mass spectrometry and accounted for

ca. 7% of the total absorbance at 214 nm. Scale-up studies allowed the isolation of significant amounts of highly

purified histone H2A (l-53)-NH,. This preparation was characterized using RP-LC, capillary zone electrophoresis,

amino acid analysis, mass spectrometty and sequence analysis using automated Edman degradation.

Keywords: Mass spectrometry; Liquid chromatography; Ion exchange; Chromatography; Electrophoresis; Histone

H2A (l-53)-NH,; Peptides

Genetic material is organized in a compact structure by the association of DNA with protein. Approximately half of these proteins are histones, which are highly basic owing to the presence of a large proportion of lysine and arginine residues. The protein H2A is one of the core histones around which the DNA is coiled [l].

It has been reported that histone H2A purified from bovine ovaries inhibited the binding of gonadotropin hormone-releasing hormone (GnRH) to rat ovarian membranes and showed anti-gonadotropic activity in cultures of rat ovarian cells [2,3]. Independently, we purified a protein from rat testes which inhibited the binding of

GnRH to bovine pituitary membranes. After ex- tensive purification, a product was identified by sodium dodecyl sulfate polyacrylamide gel electro- phoresis, and a partial sequence obtained using pulsed liquid-phase Edman degradation. These data and an amino acid analysis indicated that the purified peptide responsible for the activity may be a fragment of rat histone H2A [4]. In order to confirm the biological activity of the fragment, it was necessary to synthesize and purify rat histone H2A (l-53) which, for ease of synthesis, was amidated at its C-terminus as follows: Ac-S-G-R- G-K-Q-G-G-K-A-R-A-K-A-K-S-R-S-S-R-A-G-L- Q-F-P-V-G-R-V-H-R-L-L-R-K-G-N-Y-A-E-R-V-

0003-2670/91/$03.50 0 1991 - Elsevier Science Publishers B.V.

216

G-A-G-A-P-V-Y-L-A-A-NH,. Preliminary results were presented at the 21st European Peptide Sym- posium [5].

EXPERIMENTAL

Apparatus The instrumentation used has been described

previously [6,7]. Isolation of peptide fragments for sequence analysis after enzymatic treatment was effected on a ternary liquid chromatograph with a diode-array detector (Hewlett-Packard, Model 1090M). Ion-exchange chromatography was done on an FPLC system (Pharmacia LKB Biotechnol- ogy). The capillary electrophoresis system con- sisted of the P/ACE System 2000 (Beckman) with Chromjet integrator (Spectra-Physics).

Automatic Edman degradation was done on a pulsed liquid-phase sequencer (Model 477A) and a phenylthiohydantoin analyzer (Model 120A, all from ABI).

Mass spectra were measured on a JMS-HXllO mass spectrometer using liquid secondary ioniza- tion (Jeol).

Columns Ion-exchange columns. Precision Column 1.6/5

(5 x 0.16 cm i.d.), HR 5/5 (5.0 X 0.5 cm i.d.) and HR lO/lO (10 X 1.0 cm i.d.) columns were pre- packed with lo-pm Mono S resin (the charged group on the gel is -CH,-SO;), (Pharmacia LKB Biotechnology).

Reversed-phase columns. The analytical columns (25 X, 0.46 cm i.d.) were factory-packed with 5+m, 300 A C,, silica (Vydac) from Separations Group. Semipreparative columns (25 X 1.0 cm i.d.) were packed with the same C,, silica. For sequence analysis, enzymatic digests were run on RP-300 C, columns (100 X 2.1 mm i.d.) (Brownlee) at 40°C and a flow-rate of 210 ~1 min-‘.

C. MILLER ET AL.

Capillaries. A capillary (fused-silica) cartridge (50 cm x 75 pm i.d.) was obtained from Beckman Instruments.

Solvent system Buffers used in the chromatographic and elec-

trophoretic systems were degassed and filtered through a 0.22-pm Millipore filter. The column, buffer, flow-rate and amount loaded for each chromatographic separation are given in the figure captions. The wavelength at which the eluent was monitored is shown on the ordinate and the gradi- ent shape is indicated by a solid line.

The trifluoroacetic acid (TFA) buffer system has been described previously [8]. Solvent A was 0.1% TFA in water and solvent B was 0.1% TFA in acetonitrile-water (60 + 40).

The buffer system used for ion-exchange chro- matography was 50 mM boric acid in water or acetonitrile-water (35 + 65) as described in the figure captions. Solvent A was the borate, solvent B contained sodium chloride in solvent A. Solvents A and B were adjusted to the desired pH by addition of 2 M sodium hydroxide.

The buffer used for capillary zone electrophore- sis (CZE) was 100 mM phosphoric acid adjusted to pH 2.5 by addition of 2 M sodium hydroxide.

Enzymes Endoproteinases Glu-C and Lys-C were used

following the manufacturer’s recommendations (Boehringer-Mannheim Biochemicals).

Synthesis Synthesis was initiated on a 2,4-dimethoxy-

benzhydrylamine resin (3.0 g) [9]. Fluorenyl- methyloxycarbonyl (Fmoc) deprotection was achieved in to steps using piperidine in dimethyl- formamide (DMF) [4 and 7 min, 20% (v/v)] fol- lowed by washes with DMF, methanol and dichlo- romethane (DCM). Fmoc amino acid side-chains

Notes to Table 1: a The second entries in the columns headed mM AA, solvent/reagent and coupling time are for the recoupling steps. b 1 = DCM: 2 = DMF; 3 = DCM-DMF (ca. 1 + 1); 4 = DMSO-DMF (ca. 1 + 1). ’ A = DIC (1 eq.); B = HOBt (2 eq.). d X = 20% piperidine in DMF for 4 and 7 min; Y = 10% acetic anhydride in DCM for 15 min.

ANALYSIS OF RAT HISTONE H2A (I-53)_NH, 217

TABLE 1

Manual synthesis of histone H2A (l-53)-NH, a

Sequence Fmoc-amino acid mM AA Solvent b/ Coupling Deblock d

No. WV reagent ’ time (h)

53 Ala 3.5

52

51

50

49

48

47

46

45

44

43

42

41

40

39

38

37

36

35

34

33

32

31

30

29

28

27

26

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

9

8

Ala 3.5

Leu 3.5

Tyr(OtBu) 3.5

Val 3.5

Pro 3.5

Ala 3.5, 2.5

GUY 3.5

Ala 3.5

GUY 3.5

Val 3.5

Arg(Mtr) 3.5

Glu(OtBu) 3.5, 2.5

Ala 3.5, 2.0

Tyr(OtBu) 3.5, 2.5

Asn 3.5

GUY 3.5, 2.0

Lys(Boc) 3.5

Arg(Mtr) 3.5, 2.5

Leu 3.5

Leu 3.5

Arg(Mtr) 3.5, 2.5

His(Trt) 3.5, 3.5

Val 3.5, 2.5

Arg(Mtr) 3.5, 2.5

GUY 3.5

Val 3.5

Pro 3.5

Phe 3.5, 2.5

Gln 3.5, 2.0

Leu 3.5

GUY 3.5, 2.0

Ala 3.5

Arg (Mtr) 3.5, 2.5

Ser(OtBu) 3.5, 2.5

Ser(OtBu) 3.5

Arg (Mtr) 3.5, 2.0

Ser(OtBu) 3.5, 2.5

Lys(Boc) 3.5, 2.5

Ala 3.5

Lys( Boc) 3.5

Ala 3.5

Arg(Mtr) 3.5, 2.5

Ala 3.5, 2.5

Lys(Boc) 3.5

GUY 3.5

GUY 3.5

Gln 3.5, 2.5

Lys(Bo@ 3.5

GUY 3.5

Arg(Mtr) 3.5, 2.0

GUY 3.5

Ser(OtBu) 3.5, 2.5

l/A 2.0

l/A 0.8

l/A 1.7

3/A 1.0

3/A 1.0

3/A 2.0

l/A, l/A 1.3, 1.0

l/A 1.3

l/A 1.3

l/A 0.7

3/A 3.4

2/A 1.2

l/A, l/A 1.4, 1.5

l/A, l/A 1.2, 1.0

3/A, 3/A 1.7, 2.0

4/AB 3.0

l/A, l/A 2.0, 0.9

l/A 3.0

2/A, 2/A 4.3, 3.0

l/A 4.5

l/A 2.3

2/A, 2/A 2.0, 4.0

3/A, 2/A 2.0, 2.0

3/A, 3/A 1.17,l.O

2/A, 2/A 3.0, 1.67

l/A 0.75

3/A 2.75

3/A 3.5

l/A, l/A 3.0, 3.0

4/AB, 4/AB 4.0, 5.0

l/A 2.8

3/A, 3/A 1.4, 3.4

l/A 1.4

2/A, 2/A 1.3, 10.0

l/A, l/A 10.0

l/A 3.0

2/A, 2/A 1.3, 2.8

L/A, f/A 2.3, 2.0

l/A, l/A 2.3, 5.5

l/A 1.5

f/A 1.1

l/A 4.3

2/A, 2/A 1.4, 2.0

L/A, l/A 5.3, 4.0

l/A 5.5

l/A 1.2

l/A 3.0

4/AB, 4/AB 2.0, 2.0

l/A 1.4

l/A 3.8

2/A, 2/A 1.8, 0.8

l/A 1.5

l/A, l/A 1.5, 3.8

X X X X X X X X X X X X X X X X X X

y,x X X

y,x y,x y,x y,x X X X

y,x X X X X X X X

y,x X

X X

X X

y,x X X

X X

y,x X X X X

x9-f

218 C. MILLER ET AL.

were protected as follows: tert-butyl ether (OtBu) for tyrosine and serine, tert-butyl ester for glutamic acid and tert-butyloxycarbonyl (Boc) for lysine. All derivatives were from a commercial source (Bachem, Torrance, CA). The arginine guanidino group was protected with 4-methoxy-2,3,6-tri- methylbenzenesulfonyl (Mtr) and the histidine im- idazole nitrogen with trityl (Trt). Asparagine and glutamine side-chains were unprotected. N-Termi- nal acetylations were effected with 10% acetic anhydride in DCM. Recouplings were done as required during the manual synthesis. The manual synthesis is outlined in Table 1. Couplings were mediated by diisopropylcarbodiimide (DIC) in DCM, DMF, DMF-DCM (1 + 1) or DMF-di- methyl sulfoxide (DMSO) (1 + l), depending on the solubility of the Fmoc amino acids [lo]. The weight gain was 4.4 g.

TFA cleavage of the peptide from the resin and concomitant deprotection were first optimized on an analytical scale: peptide-resin (10 mg) was treated with DCM-TFA-thioanisole-water (49 + 40 + 10 + 1) [lo] (100 ~1) for 2, 4 and 6 h at 37” C. The peptide was precipitated and washed with 3 x 400 ~1 of tert-butyl methyl ether and solubilized in 0.1% TFA [acetonitrile-water (60 + 40)]. For the preparative-scale cleavage of the re- sin (1 g), the above amounts were increased lOO- fold and the reaction time extended to 8 h [9]. After lyophilization, crude peptide (600 mg) was obtained.

Purification

Two similar but not identical schemes were used for the purification of this material. In one instance, crude peptide (200 mg) dissolved in buffer A (pH 9.6) was applied to a Mono S lo/10 column and eluted as shown in Fig. 2c using the FPLC system. Fraction 12 was collected and ap- plied (in four runs) to a semipreparative C,, col- umn and eluted with a gradient of acetonitrile in 0.1% TFA (from 21 to 318 acetonitrile in 40 min) at a flow-rate of 2.5 ml/mm (data not shown). The main component was collected (20 ml) and half was vacuum concentrated to 1 ml for applica- tion to a Mono S 5/5 column. The peptide was eluted with a gradient of NaCl in 50 mM borate at pH 9.6 (from 0 to 1.0 M NaCl in 20 min) at a

flow-rate of 1.0 ml mini. The product was col- lected and dialyzed against water overnight and used for biological characterization.

In the second instance, crude peptide (two runs, 40 and 50 mg) was dissolved in buffer A (5 ml) and applied to a Mono S 5/5 column. The peptide was eluted with a gradient of NaCl in 50 mM borate at pH 9.6 (from 0 to 1.0 M NaCl in 27 min) at a flow-rate of 1.0 ml min-‘. The last-eluting component was collected (2 ml from each run), dialyzed against water overnight and reapplied to the same column twice and eluted under similar conditions (data not shown). Desalting was accomplished by application to a semi-preparative C,, column and elution with the volatile 0.1% TFA buffer (from 18 to 36% acetonitrile in 40 min) at 3.0 ml min-‘. This is the preparation that was further characterized.

Characterization

Analytical LC and cation-exchange chromato- graphic conditions used for the analysis of histone H2A (l-53)-NH, are given in the figure captions. For CZE analyses, purified histone preparations were dissolved (1 mg ml-‘) in 100 mM phosphate at pH 2.50 or 20 mM citrate at pH 2.55. Injection of the sample was accomplished by application of pressure for 4 s. A constant voltage (20 kV) was applied (current 104 PA for phosphate buffer, 19.5 PA for citrate buffer) and separation accom- plished within 10 min. The capillary length to the detector was 50 cm.

Several liquid secondary ionization mass spec- tra were measured by bombarding the sample dissolved directly in a glycerol-3-nitrobenzyl al- cohol (1 + 1) matrix or a glycerol-thioglycerol (1 + 1) matrix with Cs+ primary ions ( + 22 to + 25 kV). An accelerating voltage of + 10 kV was used. The spectrum shown represents the accumu- lation of five scans (see Fig. 6). The instrument was operated at a resolution of 1000 and calibrated with high-mass Cs(Cs1): peaks. An accelerating voltage scan with superposition of the m/z 5588 and 5849 Cs(CsI),+ peaks on the molecular ion region was used to verify the C-terminal amida- tion (data not shown).

Glu-C cleavage was effected in ammonium hy- drogencarbonate (0.1 M, pH 7.8, 37” C for 18 h)

ANALYSIS OF UT HISTONE H2A (I-53)-NH, 219

with an enzyme-to-substrate ratio of 1 : 50 (w/w). Lys-C cleavage was effected in sodium hydro- gencarbonate (0.1 M, pH 8.5, 37 o C for 18 h) with

an enzyme-to-substrate of 1 : 50 (w/w).

RESULTS

Synthesis

Rat histone H2A (l-53)-NH, was assembled by solid-phase peptide synthesis on a 2,4-di-

methoxybenzhydrylamine resin using Fmoc pro-

tection of the a-amino function of the amino acids [9]. Deprotection of the Fmoc group was achieved

with piperidine. Coupling of the different amino acids was mediated by DIC. Side-chains were protected using established protecting groups. As the side-chain amide group of asparagine and glutamine can be dehydrated to the corresponding nitrile during the coupling [ 111, l-hydroxybenzo- triazole hydrate (HOBt) (2 eq.) was added to the coupling reaction. N-Terminal acetylations were done with acetic anhydride in DCM when cou-

plings could not be brought to satisfactory com- pletion, even after recouplings, as monitored by

the Kaiser test [12]. The successive steps of the manual synthesis are outlined in Table 1. The weight gain at the end of the synthesis was 2.5 times the original weight of the resin. Cleavage of

the peptide was achieved by TFA in DCM in the presence of thioanisole and water as scavengers as described previously for the synthesis of y-carbox-

yglutamic acid-containing conotoxins [lo].

Purification

The crude histone appeared to be remarkably pure by RP-LC, as shown in Fig. 1. The condi- tions were those usually used in this laboratory to identify the major component (0.1% TFA [S]) and give an estimate of its overall purity. Because peptides half the size of H2A (l-53)-NH, gener- ally give profiles that are considerably more com- plex [6], the validity of this result appeared doubt- ful. Consequently, the crude material was analyzed by narrow-bore cation-exchange chromatography, which revealed that there were indeed other major

components present (see Fig. 2a). Using mass spectrometry, it was determined that the desired

product corresponded to the last-eluting compo-

-80

-80

m s

-40

-20

0 5 10 15 20 25 30 35O

Time (min)

Fig. 1. Crude histone H2A (l-53)-NH,. Load, ca. 15 pg in 10

~1; column, Vydac C,, (5 pm) (25x0.46 cm i.d.); buffers,

A = 0.1% TFA and B = 0.1% TFA [acetonitrile-water (60+

40)], with a gradient from 20 to 70% B in 30 min; flow-rate, 2.0

ml min-‘.

nent in that chromatogram (m/z 5604.8; retention time ca. 31 min). Other fractions eluting were

shown by mass spectrometry to be mixtures with both higher (m/z 5684.2; 29-min fraction) and lower (m/z 5583.5 and 5447.2; 29-min fraction) masses, or with m/z 5527.8, 5429.8 and 5292.9 (27~min fraction). Suspecting that the separation shown in Fig. 2a had not been fully optimized, it was recalled that acetonitrile had been used in the separation of peptides and proteins by size-exclu- sion chromatography [7]. The data shown in Fig.

2b demonstrate the beneficial effect of the ad- dition of acetonitrile to the buffers, and also sug- gest that the order of elution had probably not been altered. This was confirmed by isolation of the last-eluting component (UV detection at 214 nm), which corresponded to the desired product. This separation (without acetonitrile) was then scaled up to a Mono S HR 5/5 column (data not shown). The last-eluting component was collected, dialyzed and rechromatographed on a Mono S HR 5/5 column using similar conditions (data not

shown). A final RP-LC step using a C,, semipre- parative column (25 x 1.0 cm i.d.) and 0.1%

220 C. MILLER ET AL.

TFA-acetonitrile as the solvent system (data not Characterization

shown) yielded the purified histone fragment after Purified rat histone H2A (l-53)-NH, was char-

lyophilization. acterized by RP-LC (see Fig. 3). Under the condi-

2.0

z- 5 5 1 .o. 2

c

O-

t .p: E

a

I I I

0 10 I

20 30 Time (min)

0 5 10 15 20 25 30 35

Time (min)

100

80

60

m 8

40

20

,O

Fig. 2. Crude histone H2A (l-53)-NH,. (a) Load, ca. 100 pg in 125 ~1; column, Pharmacia Mono S Precision Column 1.6/5

(5.0 X 0.16 cm i.d.); buffers, A = 50 B M NaCl a gradient 0 to B in A

+ 65)] B M NaCl a gradient 0 to B in

S HR X 1.0 cm i.d.); buffers as in (a) with a gradient from 0 to 100% B in 50 min; flow-rate, 4.0 ml min-‘.

ANALYSIS OF RAT HISTONE HZA (I-53)-NH, 221

/ I I r- I 1 I I I 0 10 20 30 40 50 60

Time (min)

Fig. 2 (continued).

0.25, 100

5i 5 d 0.125-

f

G?

L o- ,

1 I I , 0 5

1 10 15 20 25

80

60

m

z

40

20

0

Time (min)

Fig. 3. Purified Histone H2A (l-53)-NH,. Load, 13 pg in 13 pl; column, Vydac C,, (5 pm) (25x0.46 cm i.d.); buffers,

A = 0.1% TFA, B = 0.1% TFA [acetonitrile-water (60+40)],

with a gradient from: 30 to 60% B in 25 min; flow-rate, 1.5 ml

min-‘.

60

m 8

40

tions used, a very sharp, symmetrical absorbance was observed for the purified product with this chromatographic system. This chromatographic trace is to be compared with that obtained under similar conditions for the crude preparation (see Fig. 1). Noteworthy is the broad base of the trace recorded for the crude preparation. It is clear that it was due to the presence of closely related impur- ities (which could be separated by cation-exchange chromatography, Fig. 2a and b) rather than to a band broadening similar to that often reported for large proteins. Similarly, cation-exchange chro- matography indicated the presence of a unique component (Fig. 4) eluting at a concentration of NaCl similar to that of the last absorbance shown in Fig. 2b.

Because these results were repeatable, showed very good resolution and recovery, and linear scale-up conditions could be derived from these analytical runs, it appears likely that this system can be used for the analysis of crude and purified peptide preparations. Optimizations of the chro- matographic conditions, however, may play a greater role than one may assume a priori. Indeed,

C. MILLER ET AL.

/ r I I I I I I

, Lo 0 5 IO 15 20 25 30 35

Time (mln)

Fig. 4. Purified histone H2A (l-53)-NH,. Load, 15 gg in 15 ~1; column, Pharmacia Mono S Precision Column, 1.6/5 (5.0

x0.16 cm i.d.); buffers, A = 50 mM boric acid [acetonitrile-

water (35 + 65)] (pH 9.7). B = 0.5 M NaCI in A, with a gradient

from 0 to 1008 B in 20 min; flow-rate, 0.1 ml min-‘.

using 50 mM borate (pH 9.7) in acetonitrile-water (35 : 65) and sodium as a counter ion, the purified histone fragment eluted sharply at a concentration of 0.5 M NaCl (see Fig. 4). Under non-optimum conditions (50 mM borate at pH 9.6 without acetonitrile, data not shown), it was observed that 1 M NaCl was required for elution of the peptide and that the peak width under these conditions was at least four times that observed when the

peptide was eluted in the presence of acetonitrile. Because both RP-LC and cation-exchange

chromatography had been used for the prepara- tive-scale purification of the preparation of rat

histone H2A (l-53)-NH,, the use of CZE as a third orthogonal analytical method was investi- gated. Under optimized conditions (phosphate buffer), this technique indicated the presence of one or more minor impurities (Fig. 5). Under less optimized conditions (citrate buffer), this impurity was not resolved from the product (see Experi- mental for details). As these impurities are likely to be present in small amounts and could not be isolated by any of the methods that lend them-

selves to scale-up, it was not possible either to

0.1

a 0 5 r5 10

Time (min)

Fig. 5. Electropherogram of purified histone H2A (l-53)-NH,.

Buffer, 100 mM phosphoric acid (pH 2.50); voltage: 20 kV

(constant); current, 104 pA; capillary, 50 cmx75 pm i.d.;

temperature, 25 o C.

isolate or to eliminate these components from the synthetic preparation or to obtain an appreciation of their structures.

Other less sensitive techniques that were used for the characterization of this preparation in- cluded amino acid analysis, which showed the

expected amino acid ratios (data not shown) after hydrolysis in 4 M methanesulfonic acid at 110 o C for 24 h, and mass spectrometry, which showed the expected molecular ion (Fig. 6). The liquid secondary ionization mass spectrum molecular ion region contained an intense intact protonated

molecular ion together with less intense peaks corresponding with loss of small neutral molecules such as H,O or HCO,H. Adducts with masses above that of the intact molecule were also ob-

served, which may correspond to retention of

either an OtBu ether, [M + 74]+, or ester-protect-

ing group, [M + lOl]+. An [M + 15]+ entity for which no explanation can be given was also pre-

TABLE

Sequence analysis of enzymatic digests

Endoproteinase Fragment Initial Repetitive

isolated yield (%) yield (%)

GIu-C 45-53 50 88

GIu-C 42-53 55 90

Lys-c 37-53 58 92

ANALYSlS OF RAT HISTONE H2A (I-53).NH,

I ‘. . . I.. . . 1. . . . . . . . . . . . . .

2500 3000 3500 4000 4500 5000 5500 61

223

20

Fig. 6. Liquid secondary ionization mass spectrum of purified histone H2A (l-53)-NH, measured using a glycerol-3-nitrobenzyl alcohol (1 + 1) matrix. Relative abundance is shown on the ordinate.

sent in the spectrum. Intense ions below 5 kilodal- ton were also observed.

Lastly, histone H2A (l-53)-NH, was treated with Glu-C and Lys-C enzymes (Table 2) to gen- erate digests that were analyzed by RP-LC. Frac- tions were collected and sequenced. Digestion with Glu-C enzyme produced three major products. Two of the fragments were subjected to sequence analysis. Non-specific cleavage (possibly due to impurities in the enzyme preparation) produced the 42-53 fragment. Lys-C digestion yielded two major fragments, one of which was also char- acterized by mass spectrometry. No preview of unexpected amino acid was observed on sequence analysis of any of the fragments isolated.

DISCUSSION

The solid-phase synthesis of several small pro- teins has been reported [13-171. In most instances chromatographic characterization (RP-LC) of the final products was either not available [13,14] or was used under non-stringent conditions [15,17]. In one instance, crystallization of the final product in poor yield and x-ray diffraction studies were

used as criteria of purity [18]. In another [17], despite the absence of negative controls, an en- zymatic activity was generated and the material shown to be homogeneous on RP-LC (in the sense that closely related impurities could not be sep- arated from each other) after one size-exclusion chromatographic step. It should be noted that some of the above data [13-15,171 do not exclude the possibility that these proteins could be less than a few percent pure.

Because mild synthetic and new chromato- graphic strategies have recently become available (Fmoc approach to solid-phase synthesis and ana- lytical cation-exchange chromatography and CZE), it was decided to apply them to the synthesis and characterization of a relatively large and repre- sentative peptide: rat histone H2A (l-53)-NH,. As shown in Table 1, except for the fact that one third of the residues needed double couplings (see column 5) either because the ninhydrin test of Kaiser et al. [12] was still positive or because coupling was to a Pro or Gln residue, which are notorious for displaying weak positives, most cou- plings were effected in ca. 2 h with a relatively small excess of acylating reagent (ca. threefold). The need for acetylation after coupling was

224 C. MILLER ET AL.

minimal and applied nine times, as shown in the last column of Table 1. From experience, the ease of assembly of the protected peptide on the resin was not significantly different from that of peptides synthesized by the Boc strategy. TFA cleavage also used standard laboratory proce- dures.

Analysis of the crude peptide by RP-LC is illustrated in Fig. 1 and indicates the presence of a unique and major component. As expected, how- ever, impurities accounting for more than 80% of the crude preparation could be separated from the desired main product by cation-exchange chro- matography. Most drastic in that separation was the effect of added acetonitrile (see Fig. 2b). Iden- tification of the desired product among all the impurities was achieved most efficiently by mass spectrometry and confirmed by amino acid analy- sis. Two sets of impurities, isolated as mixtures of at least three components each and the presence of which was recognized by mass spectrometry, were not further characterized. Further purifica- tion and desalting by RP-LC of the desired frac- tion yielded H2A (l-53)-NH, which was ulti- mately isolated with a purity probably close to or greater than 90%. This last step is particularly critical in that it allows easy concentration and desalting of the samples and selection of the coun- ter ion [6].

Despite the fact that chromatographic condi- tions that took advantage of ionic and hydro- phobic properties of the peptide mixture were used, evidence of residual amounts of impurities can still be found from a critical evaluation of the mass spectrometric data, peptide mapping, se- quencing data and CZE, a technique that unfor- tunately cannot be used preparatively as yet. Be- cause H2A (l-53)-NH, is acetylated at its N- terminus, direct sequence analysis was impossible. Sequence analysis of the C-terminal fragments isolated from Lys-C and Glu-C enzymatic digests confirmed the identity and integrity of those frag- ments. Analysis of the mass spectrometric data indicate the presence of H2A (l-53)-NH, as a major component and of entities with masses above that of H2A (l-53)-NH,. Because of the large difference in mass and therefore, charge be- tween the [M + H]+ and the ions below 5 kilodal-

ton, it is believed that they should be chromato- graphically distinct components. Therefore, the ions observed below 5 kilodalton are not consid- ered to be representative of impurities. Presuma- bly these are fragment ions. The nature of the adduct observed above the molecular mass or their amount remain to be determined.

Conclusion

It has been found that RP-LC could not be relied on as the only chromatographic step for the purification and analysis of H2A (l-53)-NH, and it is suggested that the purity of synthetic peptides larger than 30-40 residues should be documented by more than one analytical technique. Prepara- tive cation-exchange chromatography proved to be an excellent orthogonal technique for the purifica- tion of peptides and because it has been found that narrow-bore cation-exchange chromatogra- phy and CZE complement RP-LC for the de- termination of the purity of synthetic peptides, these techniques are now being used routinely in these laboratories. Similarly, mass spectrometry is used for the identification of all synthetic peptides. It should be made clear that it is not claimed that any of the techniques presented here have been optimized to their limits. It is therefore concluded that the methods presented here, when used with discrimination and critically, can considerably ex- tend the limits of RP-LC for the purification and characterization of peptides synthesized by Merri- field’s solid-phase approach.

This research was supported in part by NIH grants HD 13527 and the Hearst Foundation. The authors thank D. Pantoja for technical assistance, R. Hensley for manuscript preparation and Dr. Terry Lee of the Beckman Institute of Immunol- ogy (City of Hope, Duarte) for preliminary mass spectrometric data. The Mono S Precision Col- umn 1.6/5 was kindly provided by Pharmacia LKB Biotechnology.

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