Conformational effect on the fragmentations of peptide derivatives in field desorption mass spectrometry

Vol.

146,

No.

July

31,

1987

2, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

907-911

CONFORMATIONAL EFFECT ON THE FRAGMENTATIONS OF PEPTIDE DERIVATIVES IN FIELD DESORPTIONMASS SPECTROMFTRY Hideaki Eiji

Tsunematsu*t, Shizuko Hachiyama", Ryuichi Isobe**, Ishida*, Mayumi Kakoi", and Magobei Yamamoto"

#Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Jonan-ku, Fukuoka, 814-01, Japan **Center of Advanced Instrumental Analysis, Faculty of Pharmaceutical Sciences, Kyushu University 62, Maidashi, Higaohi-ku, Fukuoka, 812, Japan Received June 24, 1987

Fragmentations of N-benzyloxycarbonyl-protected tri-peptide ethyl esters containing proline at the P2 site were compared with those of the corresponding peptide derivatives containin no proline in field desorption mass spectrometry. The fragment ion [M-107] F due to a loss of the benzyloxy group from a molecular ion was observed in the field desorption mass spectra for the peptides containing no proline , while it was not found in the peptides containing proline at all. These results suggest that the conformational difference of the peptide derivatives attributable to the existence of proline has an effect upon fragmentations in the field desorption ionizing process. 'G1987 Academic press, 1°C.

It

is well-known

useful

that

Field

method for determining

because it

has a soft

Desorption molecular

ionizing

that

FD mass spectrometry

sequence of small peptides which organic

field

during

molecules

induced surface

(1,2).

is useful Thereare

can be converted

the FD process,

of organic

show the molecular

complex alone as the peak of highest

reported

ions

weights

process for thermolabile

compounds and the FD mass spectra cation

(FD) mass spectrometry

for example,

reactions

relative

compounds,

and nonvolatile ion or molecularintensity.

for determining several

to ions,

It was the amino acid

proposed mechanisms by ion clusters

resonance electron

and thermal

is a

decomposition

or fragment tunneling,

(3-7).

bo whom all correspondence should be addressed. Abbreviations: GPA, p-guanidino-L-phenylalanine; 2, benzyloxycarbonyl; OEt, ethyl ester; FD, Field Desorption; EI, Electron Impact; e.h.c., emitter heating current. 0006-291X/87 $1.50 907

All

Copyright (b 1987 rights qf reproduction

by Academic Press, Inc. in at1.v ,fbrm reserved.

Vol.

146,

No.

2, 1987

BIOCHEMICAL

We have synthesized ethyl

esters

the GPA specific

for

to study the subsite

of the synthetic

we noticed

the peptides

that

containing

analogs

esters

of tri-peptide

viously steric tions

of molecular

fragmentations

the existence

that

this

in peptides it

and analyzed

of N-Z-protected

tri-peptide difference

of Pro at the P2 site

the

them in

that

this

by any other

that

in the fragmenta-

paper we describe

esters

dis-

pre-

also seems possible

ones participate In this

from

was attributable

became clear

it

In addition,

the

group

synthesized fact

which has not been explained

ions in FD process.

with

by FD mass spectro-

were much different

no Pro. We therefore

than electrostatic

depend on the conformational

substrates

As a result,

proposed mechanisms (7). other

of the peptides

of the benzyloxycarbonyl

to ascertain

by FD mass spectrometry.

factors

peptide

of Pro at the P2 site

covery was a new finding

tri-peptide

enzymes. When we confirmed

Pro at the P2 site

those for the peptides

detail

COMMUNICATIONS

(GPA) or arginine

interactions

the fragmentations

containing

to the existence

RESEARCH

p-guanidino-L-phenylalanine

enzyme (8) and trypsin-like

chemicalstructures metry,

BIOPHYSICAL

the N-benzyloxycarbonyl(Z)-protected

(OEt) containing

(Arg) at the P, site

AND

that

the

in FD mass spectrometry

of the peptide

derivatives

due to

in peptides.

MATERIALS AND METHODS All amino acids used in this experiment were L-enantiomers. GPA was synthesized as described previously (9). N-benzyloxycarbonyl-protected tripeptide ethyl esters containing GPA and Arg were prepared by using dicyclohexylcarbodiimide as a coupling reagent. Syntheses of these peptide substrates will be reported in detail elsewhere. The chemical structures of the synthetic peptides were determined with the aid of FD and EI mass spectrometry and elemental analysis. All other chemicals were of analytical or reagent grade. Measurement condition of mass spectrometry The mass spectrometer employed was a JEOL JMS-DX300 double focusing model interfaced to a JEOL JMA-3500 data system (Tokyo,Japan). FD mass spectrometry: Ion source accelerating potential was 3 KV for the field anode and -5 KV for the slotted cathode plate. A dimethyl sulfoxide solution of the sample was applied to the carbon emitter. The emitter heating current (e.h.c.) was mani ulated between 15 and 22 mA, and the optimum e.h.c. for detection of [MtH] P ions was between 19 and 20 mA. In the case of the silicon emitter, the e.h.c. was between 20 and 30 mA, and the optimum e.h.c. for detection of [MtH]+ ions was between 27 and 29 mA. EI mass spectrometry: Ion source accelerating potential was 3 XV, the ionizing voltage was 30 eV and the ionizing current was 300 uA. The spectra were recorded at about 300°C for sample and 25O'C for chamber temperature. 908

Vol.

146,

No.

2, 1987

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

RESULTSAND DISCUSSION Figure

1 shows the FD mass spectra

e.h.c.(A)

and after

protonated

for Z-Gly-Leu-GPA-OEt

optimum e.h.c.(B).

molecular

In the spectrum of Fig.

in that

to this

spectrum,

molecular (Fig.

group.

When the e.h.c.

to about 20 mA, a decrease in the intensity

increase

of the fragment

detectable

were obtained

ion at m/z 447 were observed.

In contrast exhibits

ion at m/z 431 (loss of the benzyloxy

even above optimum e.h.c.(Fig.

when the silicon

for these two peptide

in which a benzylalcohol 91 is benzylcation

emitter

a

2-B). The same results

showed similar

cleavage patterns,

ion at m/z 108 is the base peak and an ion at m/z

(Fig. 3). We therefore

containing

synthesized

tri-peptide

Pro at the P2 site

ethyl

the following esters,

X,W = Ala, Val,

ence of the fragment

ion due to the loss of the benzyloxy

ion.

the peptides

This fragment containing "'

(A)

1

while

it

for

was not in those of the 555 [M + HI+

5

47

2 .E a z .-

the exist-

FD mass spectra

io7

I

some

group from the

1 ,cH~~;~-GI~~;;~;;E:

1 s

Leu, Phe) and investigated

ion was observed in all

no Pro (II),

with

two

and some not (Z-X-Pro-GPA-OEt (I),

Z-X-W-GPA-OEt (II),

molecular

group)

was used. On the other hand, EI mass

derivatives

of analogs of N-Z-protected

analogs

was

of the [M+H]+ and an

the FD mass spectrum for Z-Gly-Pro-GPA-OEt

but the fragment

was barely

kinds

a

ion peak at m/z 539 which is the base peak at optimum e.h.c.

2-A),

spectra

I-A,

ion peak at m/z 555 is the base peak and an ion at m/z

447 is formed by the loss of the benzyloxy raised

at optimum

447[M-107]+ I 0 100

447

(B)

aJ .>

555

:

z -5 cx

108

O

Fig. after

1.

I,..,., I 0

FD mass spectra optimum e.h.c.

100

200

for

300

Z-Gly-Leu-GPA-@Et

500

400

at

(A)

optimum

(m/z) e.h.c.

and

(B)

Vol.

146,

No.

BIOCHEMICAL

2, 1987

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

539

CH2C?-CC-Giy-Pro-Gi’A-CE? (1’4

0,

l

H]’

100-j

.-c

/(B)

Fig. 2. FD mass spectra for Z-Gly-Pro-GPA-OEt at (A) optimum e.h.c. and (B) after optimum e.h.c. peptides

containing

sponding peptides results

Pro (I).

in which GPA at the P, site

suggest that

at the P2 site benzyloxy

The same results

the fragment

in N-Z-protected

tri-peptide

group from the molecular

the peptides

containing

of the other fragment tected

tri-peptide

tional

difference

esters

was displaced

to Arg. These

ion.

esters

due to the loss of the ion at m/z

108

ion was weakly observed in the spectrum

It seems that

may be formed by the fission

the fragmentations

in FD mass spectrometry

of peptide

in the presence of Pro

Though the fragment

Pro (Fig. 2-A), it

ions.

in the corre-

ion was not formed

which was assigned to benzylalcohol for

were obtained

derivatives

of N-Z-pro-

depend upon the conforma-

attributable

to the existence

of

Pro.

100

olL..i’ 100

200

( ml2

)

Fig. 3. EI mass spectra for (A) Z-Gly-Leu-GPA-OEt and (B) Z-Gly-Pro-GPA-OEt 910

Vol. 146, No. 2, 1987

BIOCHEMICAL

The mechanism involved

in the fragmentation

has not yet been clearly this

elucidated.

mechanism the positive

apart

AND BIOPHYSICAL

It

electrostatic

field

and they imply that participate

steric

factors

in the fragmentations

Therefore,

some other

bond occurred

will

for

by this

in the mechanism

ones may

ion in FD process.

mechanism for the fragmentations

in FD mass spectrometry

easily

than electrostatic

of the molecular

that

bond is situated

cannot be explained other

in FD ionizing

recognized

chemical

of this

Our results

(5).

of molecules

is generally

charge localized

from the anode and the fission

RESEARCH COMMUNICATIONS

have to be considered

of the molecular

in order to explain

ion our

results. Model buildings at the P2 site

of the N-Z-protected

show that

the Z-group

of GPA and Arg. The possiblity prevent

the fission

the effect research

that

tri-peptide is brought

the basicity

of the side chains

of the guanidino

At present,

out.

Pro

of N-Z-protected

tri-peptide

to the acidic

work along these lines

group may

To discuss

of the amino acids at the PI site,

about the fragmentations

be required.

containing

so close to the side chains

of C6HFGH20-C bond cannot be ruled .

which the basic amino acids are displaced will

esters

the esters

and neutral

is in progress

in ones in our

laboratory. ACKNOWLEDGMENT

We thank Dr. M. Nakashima (Faculty of Medicine, Kyushu University) many interesting and valuable discussions regarding our research.

for

REFERENCES 1. Winkler, H. V. and Beckey, H. D.(1972) &, 391-398

Biochem. Biophys.

Res. Commun.

2. Asauti-Poku, S., Wood, G. W., and Schmidt Jr., D. E.(l975) Biomed. Mass Spectrom. 2, 121-125 3. Miiller, E. W. (1951) Z. Phys. 131, 136-142 4. Ingraham, M. G. and Gomer, R. J.(1954) J. Chem. Phys. 22, 1279-1280 5. Beckey, H. D (1964) Z. Naturforsch. 19a, 71-83 6. Beckey, H. D. (1977) "Principle of Fmd Ionization and Field Desorption Mass Spectrometry", Pergamon, London, England. 7. Beckey, H. D., Levsen, K., Rollgen, F. W. and Schulten, H. R.(1978) Surf. Sci. 70, 325-362 8. Tsunematsu, H., Mizusaki, K., Makisumi, S., Okamoto, K. and Tsunematsu, Y. (1985) Biochem. Biophys. Res. Commun. 128, 1233-1238 9. Tsunematsu, H., Nishimura, H., Mizusaki, K. and Makisumi, S. (1985) J. Biochem. 97, 617-623 911