Modification, permethylation and mass spectrometry of arginine-containing oligopeptides at the 100 nanomolar level

Vol. 45, No. 4, 1971

BIOCHEMICAL

MODIFICATION,

AND BIOPHYSICAL

PERMETHYLATION

AND

OF ARGININE-CONTAINING AT

Piet

A.

Leclercq”?,

Louis

“Institute

Received

for

August

THE

Lipid

100

C.

RESEARCH COMMUNICATIONS

MASS

SPECTROMETRY

OLIGOPEPTIDES NANOMOLAR

Smith+,

LEVEL

and

Dominic

M.

Research and +Department Baylor College of Medicine Houston, Texas 77025

Desiderio,

of

Jr.“‘*

Biochemistry

30, 1971 SUMMARY

The amino acid sequence of arginine-containing oligopeptides, including bradykinin, has been determined by mass spectrometry. The guanidino groups of the arginine residues in 100 nmoles of eptide were reacted with acetylacetone under mild conditions to produce l 8 -2-(4,6-dimethyl)pyrimidylornithine residues. I-Acetylation and N,s-permethylation then produced peptide derivatives that were suitable for vaporization. This procedure makes argininecontaining oligopeptides accessible for mass spectrometric sequence analysis. Sequence

analysis

stantially

facilitated

sodium to

be

applicable

exception

of

by

l4,2-permethylation

peptides (3),

up To

peptides to,

but

circumvent

enzymatic

arginine and

hydrazinolysis, dicarbonyl

nor

trypsin

lysine,

(c)

condensation

the

produce

with and

the

arginine appl

to group

the

N(6).

icable:

arginine

guanidino

acids,

single

residue

which

of

(2)

permethylated

peptides of

and

amino

from

are

conversion the

usual

information

approaches

to

(b)

(5),

iodide recently

the

acetylated

beyond

three

all

sub-

have

ornithine with

either by

mono-

or

compounds.

$ Present address: The Netherlands. Fellow

with or

of

including

refined

histidine

been

methyl

been of

sequence

difficulty,

hydrolysis

C-terminal

spectra

contained

neither this

and

has

utilizing

has

residues (4)

mass

spectrometry

(1)

containing

The

mass

procedure

methionine

arginine.

arginine-containing terminus

by

This

to

cysteine

*

oligopeptides

methylsulfinylmethide.

including

(a)

of

of

the

Department

Intra-Science

of

Chemistry,

Research

Foundation,

937

Eindhoven

University

1971-1975.

of

Technology,

Vol. 45, No. 4, 1971

For sation

the

sequencing

reactions

p reduced. and

BIOCHEMICAL

are

et

al.

(9)

is

possible:

(7-9)

employed

to

Thomas

et --*

(6)

pyrimidylornithine the

also

with

cleaves

methyl

termini. modified

group

the

and

silver

of

four

arginyl

We wish

to

at

the

modification

was

--et mg,

to

form

an

arginine

is

selective

(7)

al.

(7),

peptide

al.

(8)

and bond

Orn

(DMP)“.

ion

of

(the

Bacon

cleavage

utilized

undesirable

milder

resulting)

basic

quaternized

hydrazinolysis,

salt,”

a procedure

only

condensation

accomplished

oxide

residues

as of

report

by

date

and

They

catalyst.

bradykinin to

Lenard

from on

the

product Gallop

reported

both

of

sequence

the

Jj-

successfu

(lo),

and

the

permethy

c-

ation

peptides. report 100

and

which

1,2-cyclohexanedione

iodide

the

of

bonds.

mg)

is

et

“permethylat

by

conden-

derivative

Shemyakin

175-600 that

spectrometry,

1,1,3,3-tetraalkoxypropane

under

would

(l-3

the

as

Vetter-Diechtl

stated

of

This

peptides

been

mass

a unique

conditions

peptide

peptides

information

of

used.

guanidino

Permethylation argininyl

have

derivative

removed

which

such

arginine,

al

by

because

compounds

HCI.

convert

peptides

suited

reaction

alcoholic

conditions

unknown

best

B-Dicarbonyl

acetylacetone

and

of

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on

the

nmolar

methyl

modification

level,

and

using

permethylat

acetylacetone

on for

iodide/methylsulfinylmethide

the

of

arg

arginine

carbanion

for

per-

methylation.

MATERIALS The are:

commercial

Arg.HCl*H20,

Chemical

Co.,

Chemical

Co.,

hexafluoroacetylacetone,

sources

of

Nutritional Los

the

AND

METHODS

compounds

Biochemicals;

Angeles;

utilized

in

Arg-Arge2HAc

PCR,

(SOC-0490), Inc.,

Gainesville,

” Abbreviations used are: AC = acetyl, Orn(DMP) = NG-2-(4,6-dimethyl)pyrimidylornithine, acid abbrezations.

938

TFA

(M-3265), Sigma

experiments

(F2O5O),

Ac-Ser-Arg-His-Pro*HAc.H20

; bradykinin*3HAc*H20

these

Chemical

Fla.;

= trifluoroacetyl, and

sodium

the

Fox Cycle

Co.; hydride

1,1,1,5,5,5(57%

OMe = methoxy, standard amino

Vol.

45,

No.

4, 1971

dispersion

in

chemicals

were

The

mineral

the

of

ul

at

100’

by

four

paper

1.0

ml

0.5

ten

minutes.

vacua

Acyl

at ion

of

the

avoid

removed

hours.

mixture

were

meter ionizing

were

under

in

as

optimal

was

and

the

the

mixture

point,

Schiff

bases,

heated

the

NaHCO3,

heated

this

an

acetylacetone, and

10%

mixture

at

hydrolyze

with

10 ~1

complete

of

utilized

peptide,

added

reaction

permethylation

acetylated

the

conditions

was

done

additional

3,5-dimethyl-2-

aqueous

was

in

removed

(6),

first

(2).

necessary aqueous

50 111 2% Ma2C03

phase

was

dried

and under

To

for

medium

the

with

nitrogen

in

amine

prevent

5 ul the

of

The

and

acyl

bradykinin,

modified

peptide

CF3COSC2H5 hood

groups

possible

e-acetylation (12).

reacted

free

at

the

45’

C for

reaction

before. reaction

employed

with

Other

suppliers.

(8)

each

extraction

during

peptides

dried

Low

the

ether

quaternization

C2H5SH

The

to

product

ion

trifluoroacetylation

two

by

al.

appeared order

and

N,O-Permethylat

(11)

dissolved

of were

reaction

added

was

Mass.

of

--et

nmoles

In

self-condensation

Beverly,

a variety

Vetter-Diechtl

The

The

modified

was

from

CH3COCH2COCH3

hours.

was

Co.,

COMMUNICATIONS

P205.

and

migration

PI

RESEARCH

At-q.

by

20

M CH3COOH

over

To

from

chromatography.

acetylphenol, --in

Chemical

TO 100

and

BIOPHYSICAL

obtained

reported

ethanol

C for

judged

grade

modifications.

95%

AND

Ventron

Orn(DMP)

procedure

following

20

oil),

reagent

Formation The

BIOCHEMICAL

conditions

for

permethylation

previously

described

(2).

resolution

mass

sample electron

accelerating temperature

varied

were

introduction energy

voltage

spectra by was

3.5 from

70

k V. 85O

direct

e V, ion

c to

obtained probe the

source 220’

on

an

into

ionizing

LKB 9000 the

ion

current

temperature

mass source.

60 was

spectro-

@A,

2SO”

The and

the

C and

the

probe

C.

RESULTS The

mass

spectra

of

the

derivatized

Arg,

939

Arg-Arg,

Ser-&g-his-Pro,

and

Vol. 45, No. 4, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

r IlISN31NI

940

Lo 3AIltWfd

N

BIOCHEMICAL

Vol. 45, No. 4, 1971

bradykinin

AND BIOPHYSICAL

(Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg)

RESEARCH COMMUNICATIONS

are

given

in

Figures

1-4,

respectively. In

all

the

spectra.

The

genesis

spectra, These of

Frequently,

1.

The

M-COOMe

at

Figure Arg-Arg 511

--m/e

The

(M),

piperazine

is

incomplete

methylation

ion

at

loss

406

(M-164),

m/e

l-terminal due

to

Orn(DMP)

150,

of

164,

derivatized

dominate

and

178

scheme

in

at

--m/e

arginine 322,

at

the

and

M-OMe

(R). Figure

are at

given

--m/e

291

158, peaks

the

loss

of

--m/e

293

and

541

peak

at

m/e

221

is

ascribed

The

527. at

l

the

- c the

346,

IJ/~

378,

482

are

of

is

543,

accompanied

to

to

ion 556 the

the

are

318,

sequence

correspond

--m/e

the

from

291, the

diketo-

because

dipeptide,

of

respectively.

[DMP-NH-(CH2)2-

s-terminal

arginine. at

from

--m/e the

420

(M-150),

diketo-

respectively. of

571,

derivatized

668

and

6%

satellite

methylated

fragments

Ser-Arg-His-

by the

from

serine the

series

32

+

peaks

All

these

mass

units

residue.

The

C-terminus. and

to

of

(M).

peaks

H3C-iH=CH-(CH2)3-N(CH3)-DMP

CH2-CH2-CO-

263,

the

result

observed

defined

by

of

fragment

scheme

from

at

fragments

and

methanol

peaks

Fragments

and

unmethylated

dipeptide

406,

derivatized

of

and

fragmentation The

of

molecular

cleavage

332

scheme

fragments.

Corresponding

3.

130,

occurrence

diketopiperazine to

m/e

Figure

fragmentation

the

Ions

496.

(~-178).

at

265

of

fragmentation

found

and

and

R from

ions

--m/e

279

via of

sequence

lower

136,

the

to N-terminal

--m/e

spectrum

at

(R)

R-2.

is

by

corresponds

found

in

occurring

(M)

defined

of

and 392

given

and

chain

107,

in

spectrum

at --m/e

portions

mass

--m/e

scheme

ion

is

at

235

are

are

R+l2

occurring

piperazine

Pro

--m/e

the

found

--m/e

of

The

indicated

side

263.

found

CH=CH-COOCH3]+, The

is

corresponding

are

at

fragmentation

sequence

570

the

found

molecular

C-terminus

The

at

2 contains

. and

ions

and

from

are

occur

spectrum

Figure

and

ions

these

ions

The in

fragments

The the

ion

at

1.

Vol. 45, No. 4, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNJCATIONS

2

cm w -4

942

.Y

Vol. 45, No. 4, 1971

The

l

- 9

group

in

arise

also

spectrum

are up

of

side

chain

the

from

to

mass

Figure

4.

983.

The

from

E - p

of

the

of

abundant

is

not

&-Terminal

peaks

in

at

m/e the

317,

the

345,

an

aromatic

511

and

627

superimposed

--m/e is

412

ions

corresponding

to

and

235,

lower to

(13).

1 - e

indicates

peaks”

cleavage

respectively.

two

All

“sequence

of The

However

these

771,

indicating

investigation.

units

743, fragment

Phe8

No &-terminal

under

14 mass

J-terminal

lower,

corresponding

fragments

610,

the

residue

units

bradykinin

l-terminal 442,

to

residue.

at

derivatized

414,

aromatic

14 mass

ions

of

to

assigned

at

peaks the

at

corresponding

is

first

occur

is

contains

pattern

--m/e

1013

671 and 917

--m/e

satellite

present

the

bonds

at

found

fragment

Orn(DMP)g

3 also

peaks”

satellites in

there

fragments

fragmentation

cleavage

abundant

when

tetrapeptide.

--m/e

bond

methylation

and

the

at

common

Figure

are

peak

is

s-terminal

“Sequence

residue

have

found.

Phe*

and

886,

incomplete

The

methylated

eighth

peaks

case

- _6y cleavage.

the

these

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

latter

(13).

spectrum

in

resulting

are

l

the

incompletely

given

858,

cleavage

of The

BIOCHEMICAL

genesis

, the

that

the

the

lack

N-terminus

peaks.

DISCUSSION The polar

expected

usefulness

guanidino

group

mass

spectra

presented

upon

electron

impact

particularly

with

fragments.

Possible

avoided

under In

all was

difficult

to

power

of

the

TFA-bradykinin In

an

attempt

The

very to

jj

to

- c

mass

Apparently In

group

was

the

may

the prevent

considerably increase

case

pyrimidyl

of

our

permethylation

of

the the

of than further,

group

the the the

of by

the the

residue

acid of

residues, side

has

chain

been

procedure

incompletely

bradykinin,

volatile

943

intensity

the

of

lieu

arginine amino

of

@-position

volatility

modified

the

--in

demonstrated

aromatic

and

spectrum

of

been

the

of

methylation more

the

of

cleavage

conditions

moiety has

that

(6)

a superimposed

methylate.

peptides

behavior

similar

controlled

noted.

to

in

above. is

dimethylpyrimidine

quaternization

the

TFA

the

arginine

respect

cases

material

of

of

Orn(DMP)

methylated residue

electron f-group $-acetyl guanidino

(2).

is

withdrawing as

The

we1 I. derivative.

group

was

Vol. 45, No. 4, 1971

derivatized inferior

with to

The acid

BIOCHEMICAL

CF3COCH2COCF3.

those

we

have

methylation

accessible of

oligopeptides

The

mass

spectra

of

arginine

of

these

derivatives

were

presented.

dimethylpyrimidine

residues

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

derivative to

our on

general the

100

makes

technique

for

nanomolar

level.

all

acylation

natural and

amino per-

ACKNOWLEDGMENTS The authors are grateful to K. Lyman and C. Weise for technical assistance. We acknowledge financial support from the National Institutes Health (GM-13901, NIH 69-2161, RR 259, and RR 254), The Robert A. Welch Foundation (Q-125 and Q-343), and the National Science Foundation (GB-25476).

REFERENCES 1. ::

4.

E. Vilkas and E. Lederer, Tetrahedron Lett., 6, 3088 (1868). P. A. Leclercq and D. M. Desiderio, Anal. Lett., 3, 305 (1971). M. L. Polan, W. J. McMurray, S. R. Lipsky and S. Lande, Biochem. Biophys. Res. Commun., 3, 1127 (1970). P. A. Leclercq and D. M. Desiderio, Biochem. Biophys. Res. Commun., in press. P. A. White and D. M. Desiderio, Anal. Lett., 4, 141 (1971). D. W. Thomas, B. C. Das, S. D. Gkro and E. Lederer, Biochem. Biophys. Res. Commun. , 32, 519 (1968). M. M. Shemyakix Yu. A. Ovchinnikov, E. I. Vinogradova, M. Yu. Feigina, A. A. Kiryushkin, N. A. Aldanova, Yu. B. Alakhov, V. M. Lipkin and B. V. Rosinov, Experientia, 23, 428 (1967). H. Vetter-Diechtl, W. Vetter, W. Richter and K. Biemann, Experientia,

65: 7. 8.

24, 9. 10. 11. 12. 13.

340

(1968).

V. Bacon, E. Jellum, W. Patton, W. Pereira and 8. Halpern, Biochem. Biophys. Res. Commun., 37, 878 (1569). J. Lenard and P. M. Gallop, Anal. Biochem., 2, 203 (1969). E. Schrblder and K. Libke, The Peptides, Academic Press, New York, 1965, p. 213-214. E. E. Schallenberg and M. Calvin, J. Amer. Chem. Sot., z, 2779 (1955). M. M. Shemyakin, Pure Appl. Chem., u, 313 (1968).

944

of