An approach to the differentiation of leucine and isoleucine residues in EI mass spectra of peptides

Vol. 81, No. 2, 1978 March 30, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 448-454

AN APPROACH TO THE DIFFERENTIATION

OF LEUCINE AND

ISOLEUCINE RESIDUES IN EL MASS SPECTRA OF PEPTIDES(1) R. Waern and H. Falter(2) Department of Chemistry, Sudbury, Ontario. Received

January

Laurentian Canada.

University, P3E 2C6

20,1978 SUMMARY

Residues of leucine and isoleucine cannot generally be distinguished in the electron impact (EI) generated mass spectra of N-acylated peptide esters. We have obtained the mass spectra of model peptide esters containing leucine or isoleucine in various positions and trifluoroacetyl perdeutero leucine as the N-terminal blocking group. The mass spectra of the peptide derivatives show a pair of peaks as a result of the elimination from the M+ ion of neutral fragment of perdeuterated isobutene (M+-64) from the leucine side chain of the N-terminal blocking group and isobutene or butene (M+- 56) from leucine or isoleucine residues of the peptide. The ratios of the intensities of the peaks M+-56/M+-64 show considerable variation with the position of leucine or isoleucine in the peptide chain and the length of the peptide, but for peptides which are identical except for the fact that one contains leucine and the other isoleucine in a given position the ratio is always smaller for the isoleucine The differences are sufficient to distinguish containing peptide. the isomeric residues if comparison spectra are available. Mass spectrometry tool

for

is gaining

the determination

and proteins. sequence

(3)

It

is well

can be deduced

mass spectra

of N-acylated

of

ions"

"sequence

peptide

backbone

positive

charge 0 II

of

the amino acid established

from the

which

arise

Bl

NH

sequence

largely

(EI)

fragmentation

448

A3

generated

of

the

of the

- CH - C - NH AI B2

in peptides

on the basis

fragment.

A2

as a

the amino acid

A and B and retention

0006~291x/78/0812r-0448$01,00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

acceptance

impact

esters

from the

on the N-terminal O II C -

that

electron

peptide

in positions

Rl I CF3 - C - NH - CH -l-l Al

increasing

B3

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 81, No. 2, 1978

Essential

for

spectrum the

is

of

from In

ions

must

that the

in

the

the

isomeric

each other order

of

all

chains

amino

indicated

that

acids

leucine arise

amino

leucine

preferential

elimination

from

the

leucine

residue

residue.

Unfortunately

sufficient

intensity

to provide

these

important

and isoleucine

molecule

of either

called

or ethyl

a general

isobutene

the

=

H,

basis

for

of the

side

R2

the

involving case of

isoleucine.

from occur

an in

distin-

= =

chains

of

is

now commonly

rearrangement(5).

-NH-HC&RC

H for CH3

for

The rearrangement state

radical

of a neutral

in what

the leucine Because

leucine isoleucine occurs

abstraction

via

a six

membered

of a tertiary

and a secondary of the

+* I

+ R2 -CH=C-CH3

II

R2

of ions

do not

elimination

or butene

I

Rl

et

some cases

'radical

ions

fragmentation

C

CH3,

of

of Weygand

of a propyl

OH

=

fragmentation

fragmentation

these

involves

the McLaffery

Rl

additional

isomers.

The most leucine

R.

can in

further

and a methyl

isoleucine

guishing

the

and isoleucine

of type

and isoleucine, chain

The work

the

acids,with

and isoleucine

acids.

on the basis

the mass

amino

side

from

be distinguished A by the

from

leucine

the mass of the

which

of these

sequence

common twenty

to distinguish

be considered

side

al(4)

deduction

a fact

exception

differ

the

the

greater

449

carbon

carbon atom

ease with

in

which

transition atom the

in

case of

a tertiary

BIOCHEMICAL

Vol. 8 1, No. 2, 1978

hydrogen formed

atom can be abstracted from a leucine

corresponding

ion

peptides

which

more readily of

much less

important

elimination

in C-terminal

positions

differences

and isoleucine of

or isobutene

containing

isoleucine side

via

side

side

chain

chain.

as a radical

peptides

are

the entire

leucine

chain

in N-acylated

of butene

spectra

occur

the

side

in the

and isoleucine,

of mass 57, where the

the entire

reasoning

polyamino

derivatives

than

the

leucine

leucine

eliminated

(II)

than

Similar

observed

depend on the elimination

as a radical

elimination

alcohol of

ion

containing

residues

polyamino

in the spectra

is

residue.

differences

from peptides

observed

chain

that

would be more intense

the

when these

In these

expected

trifluoro-dideuteroethyl

derived

particularly

is

from an isoleucine

of 0-trimethylsilylated alcohols

it

residue

has been used to explain

(6),

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

esters

The

generally

than

the

the McLafferty

rearrangement. The nature strong

effect

on the relative

peaks as well McLafferty with

of the N-terminal

the model peptide

trifluoroacetyl blocking

group

group

of various

peaks which

On the basis

for

this

study

to affect

has a sequence

arise

of earlier

from the work

we selected

because

favorably

this

the

N-terminal

the occurrence

of

rearrangement.

While

the EI spectra

containing

peptides

shown that

they

metastable

ion

obtain

(7)

group

valylisoleucylalanine

appears

the McLafferty

However,

intensity

as such non-sequence

rearrangement.

blocking

special

of

leucine

are generally

very

can be distinguished (MI)

or collisional

techniques

and isoleucine similar

on the basis activation

and equipment

MI and CA spectra.

450

it

has been of

their

(CA) spectra are required

to

(8).

Vol. 81, No. 2, 1978

It

occurred

distinguish spectra

BIOCHEMICAL

to us that

leucine if

a suitable

internal

generally

pretable

mass spectra into

synthetic

it

might

and isoleucine

peptides

leucine

AND BiOPHYSlCAL

be possible

on the basis

standard

we decided

peptides

containing

of EI mass

in order

group

either

Since

to yield

to incorporate

blocking

to

were provided.

must be derivatized

the N-terminal

RESEARCH COMMUNICATIONS

inter-

perdeuterated

of a number of

leucine

or isoleucine.

Methods: Perdeuterated purity

98% (Merck,

acetyl

L-leucine

excess

of

anhydride

TFA [2H] L-Leu esters

(TFA lLH]

product

by the

([2H]

L-Leu)

was hydrolyzed

intermediate

glass

off

aliquots

tube

in dioxane. and peptide

N,N-dicyclohexylcarbodiimide

was filtered

capillary

and dissolved

to the N-hydroxy

by the method of Anderson

(9).

of water

conversion

employing

and transferred

acid

amino acid

essentially

was dried

a slight

to various

ester

with

with

by the addition

lyophilized

was coupled

to trifluoro-

in trifluoroacetic

succinimide

was extracted

of minimum isotopic

by reaction

anhydride

was then

dicyclohexylurea

L-Leu)

Sharp and Dome) was derivatized

trifluoroacetic

The excess and the

L-leucine

of

ether.

further

used in the

The insoluble

and the peptide

ethyl

without

et al(10).

solid

derivative

The ether

extract

purification inlet

to a

system

of

the mass

spectrometer. Mass spectra spectrometer

with

were obtained

a resolution

definition).

The sample vial

were recorded

repeatedly.

sample was recorded The source probe

temperature

temperature.

potential

is

with

of approximately was heated

The temperature indicated

spectra

slowly

and the spectra

range over

of 70 eV. 451

with

which

the

position.

somewhat higher

were obtained

mass

1000 (10% valley

in the appropriate

was maintained All

a DuPont 21-491

than

the

an ionization

Vol. 81, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Relative

Intensities

of

Selected

I

Peaks

of

Mass

Spectra

of

+ Peptide

Derivative

Ratio

Derivatives

Normalized Ratio*

z*

WcLafferty

Peptide

Probe Temperature

ratio)

1

TFA

[*HI-L-Leu-L-Leu-OCH3

.78

100%

170-190

2

TFA

[2HJ-L-Leu-L-Ile-OCH3

.47

60%

150-170

3

TFA

[2H]-L-Leu-L-Leu-Gly-OCH3

8.1

4

TFA

[2Hl-L-Leu-L-Ile-Gly-OCH3

3.9

5

TFA

[2H]-L-Leu-Gly-L-Leu-OCH3

3.5

6

TFA

t2H]-L-Leu-Gly-L-Ile-OCH3

I

TFA

[2H1-L-Leu-L-Leu-Gly-Gly-OCH3

a

TFA

[2H1-L-Leu-L-Ile-Gly-Gly-OCH3

9

TFA

12H]-L-Leu-Gly-L-Leu-Gly-OCH3

10

TFA

[2H]-L-Leu-Gly-L-Ile-Gly-OCH3

11

TFA

12H]-L-Leu-Gly-Gly-L-Leu-OCH3

12

TFA

[2H]-L-~u-Gly-Gly-L-Ile-OCH3

* See

46%

150-170

100%

.35

150-190

10%

6.3

180-200

100%

230-250

13%

170-220

12.3

100%

210-240

9.1

74%

210-240

.98

100%

210-240

.57

58%

190-210

.82

and Discussion: Table

ester

I shows various

derivatives of these

the peptide

for

of

spectra

the elimination blocking butene

from the of

mass spectra

or isoleucine

As a result

of

the peaks z

of leucine (McLafferty

452

blocking

in various

group

positions

in

rearrangement to M+ - 64 from

show peaks corresponding

chain

methyl

have been obtained.

the McLafferty

and M+ - 56 due to the side

and peptide

the TFA [%]L-Leu

of perdeuteroisobutene

group

The ratio

amino acid

contain

leucine

chain.

of these

which

derivatives

and a residue

all

170-190

text

Results

All

100%

from the common N-terminal loss

of isobutene

or isoleucine, ratio)

is

or

respectively. recorded

for

BIOCHEMICAL

Vol. 81, No. 2, 1978

Despite

each derivative. investigated involved 1.

some tentative of

relative

the McLafferty

varies

with

the length

the position For a pair that

of

of peptide

ratio

is

of

the

leucine

(McLafferty and for

in the

which

for

the

ratio)

a given

length

peptide

chain.

are identical

and the other

lower

1.

two peaks which

or isoleucine

derivatives

always

residues

may be drawn from Table

the peptide

leucine

one contains

McLafferty

number of amino acid

rearrangement

of

RESEARCH COMMUNICATIONS

number of derivatives

intensity

from

except

limited

conclusion

result

2.

the

and the restricted

The ratio

with

AND BIOPHYSICAL

isoleucine

isoleucine

the

containing

peptide. The lower indicates

the greater

is eliminated this

point

leucine the

via

It

containing

containing the

leucine

For the McLafferty

available.

twenty

for

containing

remains other

side

pair

to 100% for

ratio

of for

between

this the

10% and 74% of

derivatives.

would

serve

provided

derivatives

residues.

resulting

decreases

as the derivatives

the

to distinguish

to be seen whether

ion

each

and expressed

as a fraction

varies

chain

To emphasize

equal

normalized

both this

which

Some limitations

even from the restricted

to an inability

leucine

ratio

a given

peptides

peptide

current

the

clearly

number of compounds investigated

The total

lead

this

isoleucine

rearrangement.

of

M+- 56/M+-64

amino acid

apparent

which

derivative

containing It

hold

for

derivatives

limited

ratio

and isoleucine

will

derivative

can be seen that

isoleucine of

ease with

the McLafferty

containing

ratio.

ratio

we have set the McLafferty

isoleucine

that

McLafferty

leucine

spectra

are

relationship contain are,

the usual

however,

number compounds investigated. from the McLafferty

become larger

to measure these

453

at all

which

ions may ultimately

or with

sufficient

Vol. 81, No. 2, 1978

accuracy. only

BIOCHEMICAL

For application

one peptide

is It

corresponding

peptide

practical blocking the other in this

which

for

direction

isoleucine

generally

either

to require

leucine the

identification.

on the use of

one of which

labelled

contains

a definite

rely

RESEARCH COMMUNICATIONS

sequencing

would be impractical

method might groups,

in peptide

available

or isoleucine.

AND BlOPHYSlCAL

contained

A more

two N-terminal

labelled

or a similar

leucine

substance.

and Studies

are now in progress.

REFERENCES 1.

For a preliminary account see: H. Falter and R. Waern, London, Abstract pH-99, 59th Canadian Chemical Conference, Ontario, June 1976.

2.

To whom correspondence addressed.

3.

H. Falter in B.S. Needleman: For recent reviews see: Advanced Methods in Protein Sequence Determination, Molecular Biology, Biochemistry and Biophysics, Vol. 25, 125-148, Springer-Verlag (1977).

concerning

this

paper

should

be

H. Nau, Angew, Chem., Int. Ed. Eng., 15 No. 2, 75 (1977). P.J. Arpino and F.W. McLafferty, Determination Org. Struct. Academic Press (1976). Phys. Methods, Vol. 6, l-89, H.H. Fessel

4.

F. Weygand, A. Prox, 20B, 1169 (1965).

5.

F.W.

6.

H. Nau and K. Biemann,

7.

R.A. Org.

8.

K. Levsen, Spectrom.,

9.

F. Weygand and R. Geiger,

10.

McLafferty,

Analyt.

and K.K.

Chem. 31, Analyt.

Z. Naturforsch.

82 (1959).

Biochem.

73,

Day, H. Falter, J.P. Lehman and R.E. Chem. 38, 782 (1973). H.K. Wipf and F.W. McLafferty, 8, 117 (1974). Chem. Ber.

G.W. Anderson, J.E. Zimmerman and F.M. Chem. Sot. 86, 1939 (1964).

454

Sun,

154-178

Hamilton, Org.

(1976). J.

Mass

89, 647 (1956). Callaghan,

J.

Am.