Mapping and isolation of large peptide fragments from bovine neurophysins and biosynthetic neurophysin-containing species by high-performance liquid chromatography

Mapping and Isolation of Large Peptide Fragments from Neurophysins and Biosynthetic Neurophysin-Containing Species by High-Performance Liquid Chromatography IRWIN

M.

CHAIKEN

AND

Received

CHRISTOPHER

March

J. HOUGH

S. 1980

A peptide mapping procedure xuitahle for rapid analysis and peptide devised for the neurophysins. Tryptic fragments of performic acid-oxidized physins I and I1 were fractionated by reverse-phase high-performance tography using y-cyanopropyl-bonded from triethylammonium phosphate tions of signments

acetonitrile. of eluted

collection

of

this peptide in cell-free

mapping biosynthetic

peaks

All peaks and

buffer

Bovine

columns. Elutions were achieved to mixtures of thih buffer with

recovery was bovine neuroliquid chromausing increasing

a gradient propor-

tryptic fragments. except for dipeptides. were separated. to particular authentic neurophysin peptides were achieved determination

procedure products

of

their

to detect subpicomole wa\ demonstrated.

Peptide mapping continues to assume an important role not only in determining the primary structures of proteins but also in identifying sites of chemical modification and demonstrating the presence of protein sequences in unknown samples. In our research on the neurophysins, mapping has become essential for determining the presence of authentic amino acid sequences in cell-free translated precursors (I 2) and isolating modified peptides from photoaffinitylabeled derivatives (3,4). Existing mapping procedures developed for the sequencing of the neurophysins (5-8) have not been adequate for analyzing and isolating large peptides from small amounts of these proteins or their derivatives. For example. the four cysteic acid-containing tryptic fragments of performic acid-oxidized bovine neurophysin II. OT-12.3, and 4 of 20. 23. 13, and 10 residues, respectively (8,9). are fractionated with limited success by the conventional method of paper electrophoreG-chromatography ( I ,4,9). The large OT fragments have poor mobilities in this system, and the two peptides. OT-2 and OT-4,

amino

acid

compositions.

amount\

The

of neurophysin

Ahby use

of

peptides

are incompletely resolved. Furthermore, there is little hope of retrieval of the peptides in good yields from paper maps. A liquid chromatographic method of mapping for the simultaneous analysis and recovery of the large proteolytic fragments would solve these problems. We therefore have developed a simple peptide mapping scheme using hplc.’ The fractionation achieved is ideal for detection and isolation of the tryptic peptides of performic acid-oxidized neurophysins I and II, including the dominant large peptides. The analytical use of this system was demonstrated for detection of small (subpicomole) quantities of neurophysin peptides in cellfree translation products. MATERIALS

AND METHODS

Bovine neurophysins I and II were obtained from bovine posterior pituitary tissue ’ Abbreviations chromatography acid adjusted taining ().(I?“;

u\ed: : TEAP to pH sodium

hplc. buffer 3 with aridc.

high-performance ( IO), 0.25 triethylamine

liquid

I‘; phosphoric and

con-

12

CHAIKEN

AND

TIME (minutes) FIG. 1. Elution profiles acid-oxidized neurophysins a Zorbax-CN complished at 0 time Flow digests

rate of

physin

II.

oftrypsin I (top)

digests ofperformic and II (bottom)

on

(0.46 x 26 cm) column. Elution was acwith a linear gradient from lOOC/r TEAP to 6OV

TEAP-40%

was 0.8 mlimin. 100 fig neurophysin

acetonitrile Samples I and

at 60 min. were tryptic 200 FLL~ neuro-

(Pel-Freez Laboratories) as cited previously ( I I ). A high molecular weight neurophysincontaining translation product, defined as a probable biosynthetic precursor of neurophysin II (I), was prepared by immunoprecipitation, using anti-neurophysin II. of [“sS]Cys-containing proteins from rabbit reticulocyte lysate cell-free syntheses by procedures similar to those described previously (1). r-Val-L.-Arg and Gly-r-Arg were from Vega Biochemicals. Per-formic acid oxidation was carried out by addition of 0.5 ml performic acid (12) to each milligram protein reaction of 4°C for 3 h. subsequent dilution with water, and lyophilization. Trypsin hydrolysis at performic acid-oxidized protein was for 1.52 h at 37°C. with 2% by weight L-l-tosylamido-2-phenylethyl chloromethyl ketonemodified trypsin (Calbiochem) in 0.1 M NHaHCO:l, pH 8.6. Digests were subsequently lyophilized. Per-formic acid oxidation of the neurophysin precursor (the proceeds immunoprecipitated from a 0.2 ml

HOUGH

cell-free translation) was performed after addition of 0.5 mg of authentic neurophysin I1 as a carrier. High-performance liquid chromatography was performed on bonded y-cyanopropylsilyl columns (Zorbax-CN. 0.46 x 25 cm. from DuPont) using a Waters Associates gradient liquid chromatograph equipped with a Model 660 solvent programmer. a model U6K injector, and two Model M-6000 pumps. Samples of neurophysin digests, obtained from 50-200 pg of protein and dissolved in lo-50 ~1 TEAP buffer, were applied to the Zorbax-CN columns equilibrated with TEAP buffer and eluted, at ambient temperature, with a linear gradient from 0 to 40% acetonitrile. The elution was performed at 0.8 mumin. The gradient was started immediately upon sample introduction and proceeded for a total of 60 min. Elutions were followed continuously by monitoring uv absorbance at 2 15 nm using a Waters Model 450 variable wavelength detector. The eluate was collected either at one fraction per minute or by peak collection using a peak detector (built by the Division of Computer Research and Technology of the National Institutes of Health) to drive an LKB Redirac fraction collector. For neurophysin digests. collected peaks were lyophilized and subjected to acid hydrolysis (6 N HCI, 1 lO”C, 24 h) followed by amino acid analysis using a Beckman Model 121 M analyser. Fractions from trials with cell-free translation products were counted in Aquasol for content of ‘9, using the ‘.C channel of a Nuclear Chicago Mark III scintillation counter. Elution profiles as monitored by absorbance were recorded using an LKB Model 2210 dualchannel recorder. RESULTS

Representative for neurophysin

elution peptides

profiles obtained upon Zorbax-CN

NELIROPHYSIN

AhllNo

ACID

COMPOSITIONS

Bovine Peptide Number Pwition I” Sequence

Art? CySO,H A\P MetO, ‘l’hr S-3 GIU Pro Gly Ala Val Ile LtX TYI Phe

hplc

ACID-OXIDIZED neurophyGn

Pt*~s

BOVINC

13

MAPPING

FROM

ELUTIOY

NFLIKOPHSSINS

OF TRYPSIN 1 .\II)

Bovine

I

DK~STS

OF

11” neurophysin

II

I

7

3

J

<

I

67-93

21-43

9-18

46hh

I--X

67-86

l.Olll

I I II) 0.7 ( I I 3.4 (3)

0.x (1)

I.5 (1)

I.h(ll

I.1 III l.O,ll

I.Y\ Hi\

ZORB\X-CN

OF

PtRFoRhllC

PEP-I-IDE

21-43

3

1

\

t!

Y-IX

44-66

I-X

x7-93

0.9 ( I)

I.3 (II

1.u (II 3.7 (5) 3.2 Ii)

2.0 2.9 3.1 2.5

11, (3) (31 I?)

5.6 (6) I.5 II) 0.5 III

I.0 II) 2.6 (41 I.1 (11 I.0 (1) I.1 (1) 2.8 (2)

0.9111

0.9 I I) 4.2 (4) 2.0 (2) 0.9 III 1.0(I) 2.0(l)

I.9 (2) 3.6 (3)

2.: 13) 4.4 I51

2.x (3) 4.5 (5)

1.9 (3) 4.0 (4)

2.6 13) 4.2 I41

I.1 II) I.1 (II 1.1 (?I

I.1 II) a.9 (I) I.1 (II

1.x 12)

0.‘) c I, 0.6,

0.9 1 I)

I.1 (II I.2 (II

0.‘) , I)

I)

I.8 (2)

” Neurophysin 1 peptides (I through 6. accounting for Y I of the 95 total reuduesl

0.‘) I I) 5. accounting are from peak\

for Yl of the of corresponding

93 total protein t&due\) and II peptide\ (I through number in Fig. I. Peptide numbering is according 10

the notation of Wuu and Crumm (91. with the correcponding sequence position\ of the values given can be compared to the values. in parentheses. representing the integral the particular authentic tryptic peptides. CySO., (cyst&c acid) and MetSO, (methionine products of Cys and Met. rupectively. No correctionr were made for destruction ‘l‘hew analysr\ repretrnt a single determination uhich ha\ heen verified hy at lea\t hplc runs.

reverse-phase liquid chromatography are shown in Fig. I. As indicated, separation of several uv-adsorbing components is effected for both protein species. The absolute positions of these peaks in the elution profiles remain fairly constant if eluted from the same column. However, a decrease in retention times has been observed with column aging.

Amino acid compositions of the eluted hplc peaks shown in Fig. 1 were obtained and compared with the compositions of the known tryptic fragments of performic acidoxidized neurophysins (8.9,13). These com-

peptides denoted. Experimental rtxidue numhers of each residue expected for wlfone) are the performic acid oxidation or +IW release during acid hydroly\i\. two additional analyw\ of two wparatr

positions are given in Table 1, along with the compositions expected for the appropriate authentic peptides. The resultant assignments of hplc peaks are indicated in both Fig. 1 and Table 1 using the notation of Wuu and Crumm (9). As expected from sequence homology, both neurophysins I and II yield fragments OT-2 and -4 of identical mobility. In contrast, neurophysin I yields OT-1, -3, and -5 of mobilities different from those for neurophysin II, and no OT-6.7. The absence of OT-6.7 and differences in OT-I and -5 mobility for neurophysins I versus II are fully expected from the substantial sequence differences of these two proteins in the amino- and carboxyl-terminal regions. More strikingly. the difference in mobilities of

14

CHAIKEN TABLE

RFrENrlONs

OF TRYPTIC

ACID-OXIDIZED

P~PTIDES

OF PERFORMIC

BOVINE NEUROPHYSINS ON ZORBAY-CN”

Neurophysin

I. X ’

OT- I

7.8

OT-2

II. X ’

laverage 5.7 (S.3:

A’.

range)

5.1-5.3)

OT-3

IO.7 4.4

OT-4

6.4

6.2 (6.2:

OT-5 OT-6 OT-8 OT-9

2.2 -

I.1 (1.1: 1.0~12) 2.2 (7. I. I.%‘.?) 0 0

0

‘I For

OT-I

calculated expression

through

6, values

from the retention X’ = [(retention

volume expected

time)]/(void dipeptides

neurophysin

II) and

both neurophysin commercial peptides parentheses X ‘, followed period

10.7 (10.7: 10.43.6 (3.6: 3.6-3.6)

of

10.8)

6.0-6.3)

of X’ (retention)

times in Fig. time of peptide)

were I, by the ~ (void

volume time). OT-8 (Arg-Val.

Values residues

for the Y4-Y5 of

OT-9

residues

19-20

(Gly-

I and II) of these

for neurophysin by the range,

for

Arg.

of

were determined using sequences. Numbers in II represent four elutions

the average run over

HOUGH

As shown by Fig. I, there are a few observed peaks which do not correspond to any authentic neurophysin peptide. The peaks eluting at 43.5 min in Fig. 1 do contain significant amino acid material (as judged by amino acid content) but their compositions do not correspond directly to any particular neurophysin-specific sequence. They could represent peptides from nonneurophysin contaminants. incompletely digested neurophysin fragments. or a mixture of these.

2

Neurophysin Peptide

AND

a

I month.

OT-3 from 1 and II arise from a single residue difference at position 9 (which is Thr in 1 versus Gln in II). Interestingly, the amino acid composition of the peak at 12 min for neurophysin II indicates the presence here of OT-6 (with Val at position 89). OT-7, the OT-6-related fragment with Be at position 89 (also reported for neurophysin II and attributed to microheterogeneity (9)). was not detected. Of the possible peptides from neurophysins I (93 residues) and II (95 residues), only two, both dipeptides, were not resolved in the Zorbax-CN hplc maps. Both of these (OT-8, Arg-Val, and -9. Gly-Arg) occur in neurophysin II, while only one (OT-9) occurs in neurophysin I. From elutions carried out with commercial peptides of these sequences as standards, both of these dipeptides elute in the breakthrough fraction of the elution. Except for these. all of the neurophysin sequences are accounted for as peptides separated on Zorbax-CN.

As a means of defining neurophysin peptide elutions on a normalized basis, retention times were related to void volume times by the parameter of retention, X’, equal to [(retention time) - (void volume time)]/(void volume time). Values of I\‘. calculated from the peak positions in Fig. I, are listed in Table 2. This table also includes, for neurophysin I, an indication of variability in observed peptide elution, as range of h-’ values determined for elutions performed over a period of a month and average X’ value from these repetitive runs. The values of L’ in Table 2 reflect the extents of retention of each peptide on the particular hplc support (here Zorbax-CN, column No. 1804).

In order to demonstrate the effectiveness of hplc mapping to detect small amounts of neurophysin-related sequences. a map was obtained for the ““S-containing tryptic peptides derived from neurophysin-containing cell-free translation products. The elution profile of label is shown in Fig. 2, along with that of an internal control of the authentic neurophysin II peptides observed by monitoring A.,,5nm. The correspondence of the major “‘S-containing components in the translation product to the elution positions of authentic neurophysin I1 peptides is consistent with previous data from pep-

NEUROPHYSIN

tide mapping on paper and establishes clearly the identity of this species as a high molecular weight neurophysin-containing biosynthetic precursor. Of note. the labeled peptide corresponding to OT-2. which could not be detected in cell-free translation products on paper (I ,?), is detected clearly in the hplc map. This observation, importantly, completes the detection of all four expected cysteic acidcontaining peptides in the high molecular weight translation product. Aside from the large early peak in Fig. 3. no major ‘%-containing peaks other than those definable as neurophysin peptides are distinguishable in the hplc maps obtained so far from translation products. Nonetheless, the possibility cannot be excluded that either the early peak or the background are obscuring other discrete radioactive components present in the digests. DISCUSSION The evolution of hplc fractionation into a technology suitable for peptide isolation and mapping is apparent (10,14-16). The use of this approach for the neurophysins offers a method of peptide detection and preparation far simpler and more reproducible than any used previously for this protein. Neurophysin peptide mapping is in some ways particularly difficult to achieve, due to the limited sites available for the most specific peptide bond cleavage agents, such as trypsin, chymotrypsin, and cyanogen bromide. The data of Fig. 1 indicate that a one-step hplc elution can achieve separations of large peptides more effectively than in any previously used methods. including multistep procedures. The unique hplc retentions of homologous bovine neurophysin I and II peptides such as OT-I or -3 are in contrast to the overlapping migrations of those peptides in two-dimensional electrophoresis-chromatography mapping on paper (1.2,4,9). The only peptides not resolved in the present work are dipeptides. which migrate largely unretarded on Zorbax-

PEPTIDE

MAPPING

IS

200

rIx 4 .2 g E ,a

100

10

FIG.

2.

Elution

20

30

40

Time

of elut~on

lmr

protile

of

50 I

anti-neurophyain

II-

recognized biosynthetic product, from cell-free translation of bovine hypothalamic mRNA in the presence of [:“S]Cys, as defined the physin sample

four

on in

authentic

trypsin

which added

of

Elution Fig. I:

Cys-containing

II tryptic fragments was derived from

tsee text). in physin 11 was and

Zorbax-CN. the legend

conditions the positions oxidized

are indicated. a 700-~l cell-free

0.5 mg authentic prior to performic

were of neuro-

The &ted translation bovine neuruacid oxidation

digestion.

CN. Collection of the breakthrough peaks from the Zorbax-CN elution and rechromatography on paper (4,6) or perhaps another hplc support would seem an appropriate way to proceed to isolate the dipeptides if desired. It is. however, the detection and isolation of the larger peptides, which make up 98 and 96% of the neurophysin I and II sequences, respectively, that pose the greatest problems. particularly for cell-free translation products. These problems appear to be solved by the present hplc method. Taken with previous results (10.17). the data here emphasize that y-cyanopropylbonded hplc columns can be a powerful mapping and isolation tool for large peptides in general. This is clearly the case for the neurophysins. given the known sequence homologies within this class of proteins (5). The major weaknesses encountered here were the lack of resolution of very small peptides and the progressive degeneration of retention times and resolution with column aging. The latter problem

16

CHAIKEN

AND

could make it difficult to compare maps obtained over a long period of time, although comparison of/i’ values or inclusion of an internal standard should allow for elution normalization. Overall. the clear-cut advantages of high sensitivity and accuracy in analytical mapping and the ability to recover peptides emphasize the considerable appeal and utility of the hplc method.

HOUGH Hormone affinity

We

thank

Dr.

Pamela

Bridgen

the use of the high-performance instrument and for her helpful the course of this work. We Seeman

for

his

help

her

guidance

amino

and Paul their useful

acid

I lY7l)

10. I I.

343-366. Fischer.

C..

and

Chaiken,

Giudice.

L. C.,

3.

clrc’l?l. Klausner,

254, I 1?611 1770. Y. S., McCormick.

I. M. X2-90. 4.

McCormicL,

(1978)

and

[/!I. W.

./.

M. (1979)

A.,

Curd.

.I. E..

/l,l~lrrrtr~lc~/rc~l~~;,\i~~~

Hirs. C. (Hirr.

14.

Hansen.

J.,

M.

(1979)

Yrr~c.

IS.

I. M. ( 1979)

J. Rio/. 16.

W. M.,

and

Pr/~tidr

Pro/c,i/r

An

Investigation

11,

of the

17.

W. Heal-n,

S.. Bishop. M. T. W

C‘hctrr.

McMillan, D. B.. ~f.c~/rtlot/~

254.

and

M.. and 277,

Bi~~c~l~c,m.

I. M.

York. J.. and

in Enzymology pp. lY7~lYY.

Acher.

I‘..

R. (IY75)

Currie.

B.

I,..

K. ( lY77)./.

C. ,4., Prestige, R. I... (lY78) ,d,rtr/. Llir~c/rc,f?r. R.

H.

(lY7Y)

12.

Cecka. J. M.. Hood, L.. McDevitt. H. 0. 11979) 663-665.

1.

Chaiken.

I I,

Wasserman, 710X-71-

Walter,

14. 595-602.

Grcibrokk.

89, 203-212. Fullmer. C. S.. and Rio/.

Chaiken,

Rec.

Hancock. and

R.

634-639. c‘it~~~,,t~ii,~~r.

H. W. (1967) i/t Methods C. H. W.. ed.). Vol.

J.

Acher,

S. E. (1976)

Johansson, K. N.-G.. and Folkers, C‘/?~~~/mrr,~~v~. 135, ITS- 164. I.

Chaiken,

Crumm.

.Academic Press, New Chauvet. M.-T.. Chauvet. FEBS l.vrr. 58. 234-7-37.

Ntrr. Ac,trd. SC,;. USA 76, 3800-3804. 2.

and

J.. and 475-485.

69.

Audhya. T. K.. and C‘/~C~/,I. 253, 5019-5014.

13.

REFERENCES L.

IO, 6043-6063.

Kc,.\. (‘o~~/~//~I. 68, E. (197X) .I. Liq/lit/

E.

(1977)

and on

D. H.. ./. 61iol.

‘I.-C..

Bio/‘h\. Rivier. J.

17.

(1/1C1?2.

M.-T.. Chauvet. E//r. .I. Bi,~c.llr,/~r.

X. Schlesinger. R. ( 197X)

manuscript.

1. Giudice.

./. Hir,/.

7. Chauvet. ( 1976,

analy-

Hargrave comments

by PhotoUniversity of

Maryland.

III

liquid chromatography suggestions throughout also thank Mr. Jonathan

in performing

ses and Drs. Akira Komoriya Mr. David Abercrombie for thix

for

Site of Neurophysin Ph.D. Thehi>.

5, Breslow, E. ( 1979) Atrnu. RvL,. Ri~~~/rc~t~r. 48. ZCI374. and reference\ %itcd therein. 6. Wuu, T-C., Crumm. S. E.. and Saffran, M.

Y. Wuu.

ACKNOWLEDGMENTS

Binding Labeling.

Murphy. ,Vtrrrrfc,

./.