Peptide sequencing using the combination of Edman degradation, carboxypeptidase digestion and Fast Atom Bombardment mass spectrometry

Vol. 104, No. 4, 1982 February 26, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Pages 1223-1230

PEPTIDE SEQUENCING USING THE COMBINATION OF EDMAN DEGRADATION, CARBOXYPEPTIDASE DIGESTION AND FAST ATOM BOMBARDMENTMASS SPECTROMETRY Carol Chemical

University

and Dudley Lensfield

H. Williams Road, Cambridge,

Michael R. Hanley Pharmacology Unit, Medical Cambridge

Neurochemical

Received

V. Bradley Laboratory,

November

23,

Research

Centre,

CB2 1EW

Hills

Road

1981

It is shown that the application of Fast Atom Bombardment (FAB) mass spectrometry to the sequence determination of peptides can be aided if the technique is used in conjunction with systemBoth the Edman degradation and atic cleavage of the peptides. carboxypeptidase digestion have been successfully applied in the sequence analysis of model synthetic peptides and mixtures of such peptides. Introduction Peptide of N-acetyl special

sequencing permethyl

advantages

N-terminally (lo-30

containing sequenced

by this

the

ability

size;

studied

to

study

with

are routinely

1,2

required

residues

and has some

in the study

mixtures.

Small

of amounts

and peptides

can be conveniently

of FAB mass spectrometry technique

for

to

study

peptide

sequencing

peptides; larger

4 and 20 amino acid

our present

by Barber

advantages

underivatized

facility

between

mass spectrometry

established

sequencing

and their

The most significant

and the

containing

peptides

(EI)

technique.

another

spectrometry.

is well

4 and 9 amino acid

The introduction has provided

impact

conventional

of peptide

between

electron

derivatives

over

blocked

nanomoles)

using

et al3

by mass

of this

method are

a reduction

in sample

peptides.

4s

residues

are conveniently

Peptides

equipment. 0006-291X/82/041223-08$01.00/0 1223

Copyright 0 1982 by Academic Press, Inc. All nghrs of reproduciion in any form reserved.

BIOCHEMICAL

Vol. 104, No. 4, 1982

A sample size sufficient

for

of

approximately

molecular

weight

or negative

ion mode, but

amino acids

to be determined.

between

both

are observed

complete

sequence

and negative

of sequence

is clearly

(>50 nanomoles) for

not

often

some peptides.

and the allow

peptide

the complete

EI mass spectrometry mass spectrometry6 sample sizes nanomoles

of generating digestion removal

to generate

reported

Materials

molecular

a mixture

and their

varies

dependent since

is

considerably. on sample size

large

extensive

sample sizes

sequence

frequently

sequence

peptides

For weight

information

with

This

the

in the positive

information

not ions

complete

does not

complements

from the disadvantage

amino acid

alternative.

model peptides

factor

required.

coupled

may enable

determination.

suffers

high

of N-terminal

mising

sample sizes peptides,

residues

via

larger

less

than

fragments, the

preliminary

methods

and chemica

subtractive offer

has been successfully

mixtures,and

a pro-

applied

results

are

and Methods peptides

San Carlos,

were obtained

California. 1224

20

Carboxypeptidase

FAB mass spectrometry

strategy

that

alternative

are desirable. of C-terminal

FAB

here.

Synthetic Inc.,

but

sequence

Edman degradation,

in part

of derivatized

are usually

and for

is

C or N terminal

sequence

and this

of peptides

to provide

either

Sequ-

ion modes from

available

Sequence information

absence of

information.

to be determined.

the only

fail

sequence of

typically

and negative

information

ions

the positive

the

sample sizes,

of the peptide

sequence

allow

some sequence

ion FAB mass spectra

The abundance but this

Larger

is generally

in either

normally

in the positive

of the

The amount of

determination

afford

the N- and C-termini

RESEARCH COMMUNICATIONS

0.1 nanomoles

does not

1 and 5 nanomoles,

ence ions

AND BIOPHYSICAL

from Peninsula

Laboratories

to

BIOCHEMICAL

Vol. 104, No. 4, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Edman Degradation: Various modifications of the Edman reaction sequence were investigated. The most successful method for combination with FAB mass spectrometry appeared to be the following: 1. Coupling: 5-10 nmol of peptide were dissolved in 200~1 of 50% aqueous pyridine. 100~1 of 5% phenylisothiocyanate in pyridine were added and purged with oxygen-free nitrogen, at 37OC for 1 hour. The solution was capped, and incubated extracted twice with heptane:ethyl acetate 2:l v/v and lyopholized. 2. Cleavage: 150~1 of trifloroacetic acid were added to the above product, the solution purged with oxygen-free nitrogen, at 37OC for 30 minutes, and then lyopholized. capped, incubated 3. Extraction: The lyopholized product was suspended in 1 ml of distilled water and the phenvlthiocarbodimide amino acid derivative extracted into l-ml of n-butyl acetate. The aqueous layer was lyopholized and the product redissolved in 200~1 of 50% pyridine. 40 ~1 (l-2 nmol of peptide) were withdrawn, lyopholized and analysed by FAB mass spectrometry. The remaining solution was subjected to the next cycle of degradation. Carboxypeptidase Y Digestion: The peptide mixture (ca. 10 nmol) was digested using carboxypeptidase Y from Bakers Yeast (Sigma Chemical Company) with a 5O:l molar ratio of substrate to enzyme in 1 ml of distilled water. The solution was incubated at 37OC and samples (ca. 2 nmol) were withdrawn at 2,4,6 and 18 hour intervals, and the mixture of products isolated by lyopholization.

Results

and Discussion

Figure molecular

1 shows the positive ion regions

Edman degradation LYS

Pro

of peptides

l(a)

and the

generated

of nona-substance Gln

IA--I-AI 965 868 Figure

ion FAB mass spectra

Gln 740

shows a mixture

sodium salt

Phe

Phe

at -m/z 1094 and the

octa-peptide

have the

Met

NH2 -1

of the

of

glutamine

GUY Leu

612

The mass difference

an N-terminal

subtractive

P (1).

protonated

m/z 988 obtained --

indicates

by the

of the

128 between

lysine

same nominal

after the

intact

generated or glutamine

molecular the

first

ion m/z 966 --

Edman cycle.

nona-substance by Edman degradation residue.

(Lysine

mass but may be distinguished 1235

P MH+

and by

Vol. 104, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MNO+ 891

MH* 741

MN2 763

Figure

1:

acetylation

The positive ion FAB mass spectra of the molecular ion regions of peptides generated by the subtractive Edman degradation of nona-substance P.

of

heptapeptide in Figure

former).

generated l(b).

sodium salt

The molecular

after

The protonated

The ions

ated molecular

Edman cycles

residues

the

of

are reproduced

ions

for

ions

of the is

reproduced

ion at -m/z 869 and the

second N-terminal

form

amino acid

907 are due to the (oxidation

proton-

of methionine)

respectively.

the

the

products

in Figures

hexapeptide

741 and 613 respectively. molecular

molecular

ion of the oxidized

sodium salt,

ion region

second Edman cycle

at -m/z 885 and m/z --

The FAB mass spectra

molecular

the

at -m/z 891 establish

as proline.

and the

the

establish

as two glutamines.

of the third

and fourth

and l(d).

Protonated

l(c)

and pentapeptide

Mass differences the

third

and fourth

In the positive 1226

of

occur

128 between N-terminal

ion

spectrum

at m/z -the amino acid of

the

Vol. 104, No. 4, 1982

pentapeptide,

Figure

to the product amino acid

BIOCHEMICAL

l(d),

obtained

residues

Edman degradation

size

of -ca.

RESEARCH COMMUNICATIONS

at m/z 629 is again -of methionine.

However,

the

fourth

in principle

of

further

of the

cycle

N-terminal

has been achieved

with

by the

sample

allowing

to observe

degree

unambiguous

of N-terminal amenable mixture

residues.

to the

study

containing

of simple

5 nanomoles

Ll965

cs

r

706

578

Gln

mixtures. of both

presented

For example, eledoisin-related

assignment here

is

is

a synthetic peptide

P (2) was subjected

to the Edman degradation.

Ile

NH2

Gly

Leu

Met

Phe

Phe

GUY Leu

ion FAB mass spectrum reproduced

in Figure

and associated

Met

NH2

from structure

related

peptide

Both visible

the

protonated (in

metal

generated

-2 produced

the

salts

same spectrum

I227

the

first

molecular

at -m/z 891 and

by removal

of N-terminal

of the eledoisin-

a pentapeptide

molecular

and 907) at -m/z 579 and 601 respectively

after

The protonated

Edman degradation

2.

structure

obtained

2.

alkali

heptapeptide

proline

are clearly

strategy

a high

-3

907 are due to the

578.

for

the

868

ion at -m/z 869,

weight

the

requires

all

of

2 -

Gln

The positive Edman cycle

peptide

However,

(2) and octa-substance

Pro

original

the

FAB mass spectrometry

although it is not always possible 5 components. The classical Edman degradation of the

sample

determinations.

mixtures,

of purity

four

subtrac-

on a total

a proportion

degradation

attributed

The first

been determined

and FAB mass spectrometry

after

Some success

ion

by oxidation

10 nanomoles.

remained

possibility

the

have therefore

tive

still

AND BIOPHYSICAL

of molecular

ion and the that (Figure

gives 2).

sodium salt

m/z 869, --

891

Vol. 104, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I

MH+ 579

MNa*

Figure

2:

The positive ion FAB mass spectrum of peptides from eledoisin-related peptide and octa-substance after the first Edman cycle.

Subtractive

Edman degradation

fragments,

for

successful

presumably

organic for

example

extraction

peptides

used in this unable

success

of C-terminal sequence using

peptides

C-termini,

subtractive

the

were less

information

carboxypeptidase

in the

noticeable for

substance

example

the

P related

peptides

Edman degradation

was

information.

use of carboxypeptidase

peptide

1228

peptide

are more soluble

was particularly

sequence

problem,

on shorter

and tripeptides,

common to the

Hence the

this

C-terminal obtained

This

C-terminal

mixtures

was

small

hydrophobic

sequence

study.

To overcome

determine

since

containing

to provide

generate

tetrapeptides

solvents.

X-Gly-Leu-Met-NH2

studies

generated P

fragments,

and hence to

was investigated. Y to

to

digest

Some a mixture

Vol. 104, No. 4, 1982

of

BIOCHEMICAL

5 nmol eledoisin-related

variation

of the

enable

the

example,

ion of

peptide

enzyme:substrate

observation

analysis

essentially

AND BIOPHYSICAL

of

of the

unchanged

all

and octa-substance ratio

C-terminal

2 hour digestion

octa-substance

time

However, did not

fragments.

For

sample by FAB showed

P and a small

peptide

P.

and reaction

possible

the eledoisin-related

RESEARCH COMMUNICATIONS

to give

the

amount of digestdigestion

products

-a and -b shown in -4. Phe

LYS

b

C

Six

hour digestion

octa-substance products

fragment

a large

amount of unreacted

no intact

digestion

Gln

study

Phe

it

digestion

would

provide

differing

rates

for

observation

GUY Leu

However no additiona

Met

that

C-terminal

sequencg since

C-terminal

peptide

of

C-terminal

fragments.

all

reduces

the possible

NH2

carboxypeptidase

cleaving

and often

determined,

provides

some

C-terminal

by two amino acid

sequence

limiting

the possible 1229

However,

compositions.

are often

combinations.

the

information,

amino acid

residues

the

bonds do not

sum of the masses of two amino acid

thus

obtained

-5

unlikely

a complete

generally

and hence the

peptide

--I d

appears

mixture

differing

and an

P and a small

peptide.

Phe

digestion

Products

products

octa-substance

I

the

P, 2,

from eledoisin-related

e

allow

digestion

were observed.

Gln

From this

of undigested

However,

octa-substance

eledoisin-related

products Pro

for

carboxypeptidase

revealed

proportion

peptide.

-e was generated

of the

18 hours

digestion

-4

P and eledoisin-related

Analysis

after

indicated

a and e were observed

additional --4

again

a

observed

residues

may be

Vol. 104, No. 4, 1982

It

is

digestion

concluded

that

offers

ation

from peptides.

AND BIOPHYSICAL

the

and subtractive

metry

to small

BIOCHEMICAL

combination

of

Edman degradation

a promising

strategy

carboxypeptidase with

FAB mass spectro-

from obtaining

The combination

sample sizes

RESEARCH COMMUNICATIONS

and extensive

sequence

of techniques purification

inform-

is applicable

of peptides

is

unneccessary. References 1.

Morris, H-R., Williams, J ., 125, 189-201.

2.

Morris,

3.

Barber, M., Bordoli, (1981) Nature, 293,

4.

Williams, Biochem.

5.

Morris, H.R., Panico, M., Barber, M., Bordoli, R.D., and Tyler, A.N., (1981) Biochem. Biophys. 101, 623-631.

6.

Williams, D.H., Bojesen, G., Auffret, (1981) FEBS. Letts., 128, 37-39.

H.R.,

(1980)

D-H., Nature,

and Ambler, 286,

C.V.,

1230

(1971)

Biochem.

447-452.

R-S., Sedgwick, 270-275.

D.H., Bradley, J., in press.

R.P.,

R-D.,

Santikarn,

A.D.,

and Tyler, S.,

A.N.,

and Bojesen,

G.,

R.S., Sedgwick, Res. Comm.,

and Taylor,

L.C.E.,