Structural analysis of human hemoglobin variants by mass spectrometry

International

Journal

Elsetier Scientific

STRUCTURAL

of Mass Spectrometry

Publishing Company,

ANALYSIS

OF HUMAN

48 (1983)

and ion Physics, Amsterdam

-

HEMOGLOBIN

Printed in The Netherlands

VARIANTS

Y. WADA' , A. HAYASHI' , T. FLJJITA' , T. MATSUO', 'Osaka

Medical

840 Murodo, 2

Institute

Center

Izumi,

and Research

Osaka

of Physics,

'Department

of Physics,

I-I Machikaneyama,

590-02

Institute

BY MASS

SPECTROMETRY

KATAKUSE3

I.

for Maternal

and H. MATSUDA2

and Child

Health,

(Japan)

College

of General

Faculty

of Science,

Toyonaka,

209

209-212

Osaka

Education, Osaka

Osaka

University

and

University,

560 (Japan)

ABSTRACT FD and FAB mass spectrometries were applied to structural analysis of human hemoglobin variants. All peptides were identified in the FD mass spectrum of the tryptic peptide mixture of 8-globin. The method permits us to detect the abnormal and moreover to determine the type peptide containing amino acid substitution, and position of substitution without further conventional amino acid analysis.

INTRODUCTION Human hemoglobin in a globin structures graphy Since

polypeptide. involve

and/or

peptide

are usually

Conventional

separation

weight

we adopted

the biochemical

acid

caused

by single

procedure

of tryptic

electrophoresis

and amino

molecular

metry.

variants

amino

for analysing

peptides

(peptide-mapping

their

of abnormal

technique),

acid

substitution primary

globin

by chromato-

detection

of abnormal

analysis.(ref.l) of peptide

can be determined

it to the structural

procedures

above

analysis

by FD or FAB mass

of abnormal

globins

spectro-

instead

of

mentioned.

EXPERIMENTAL Sample

preparation

Normal Human

and two abnormal

B-globin

established. of valine causes patient

for glutamic

sickle

cell

acid

disease.

with a history

Another this

Normal

globins

OOZO-7381/83/000+0000j$03.00

acids

globins

was

abnormal

were 0

and

digested

which

is completely has a substitution

from the N-terminal

globin

hemoglobin

chromatography.(ref.l)

and the sequence

was

(Bx-globin)

we were

globin

by coIumn

Bs-Slobin,

at the 6th position

Since

B-gIobin.

prepared

of 146 amino

of the patient,

was not. These

were

of polycythemia

strategy.

counterpart

BX- and normal globin

is composed

One of the abnormal

any analysing normal

globins

and

could

the

B-globin

not be separated to analyse

Bx-globin

were

by trypsin

from

a

it has been withstanding

compelled

1983 ElsevierScientific

end and

from the

the mixture

aminoethylated,

for I hour and

PublishingCompany

of but BS-

lyophilysed.

210 Mass

SPeCtrometrY

FD mass meter.

spectra

were

obtained

A few pg of sample

silicon

emitter

(JEOL CO.

was dissolved

by a microsyringe.

Ltd.).

line(JEOL

type double

in 50% methanol

FAB mass

spectra

A half mg of sample was dissolved

and then a drop of glycerin of 3 keV. Mass

with Matsuda

data were

was added.

analysed

Ionization

by JMA2000

focusing

mass

and then transfered

were obtained in water

was

data

to a

with JMS-DX300

on the sample

performed

or JMA3100

spectro-

by Xenon

analysis

holder ion beam

system

on-

Co. Ltd.).

RESULTS FD and FAB mass Fig.1.

In FD mass

molecular detected

detected

of tryptic

as doubly

dehydrated

protonated

ions([M-l]-

but other

mixture

peptides

ions [M+Z]++.(ref.2) for example

are shown

be detected

in

as protonated

Some peptides

were

In FAB mass spectrum, FAB) of several

T-l(M=951),

was not detected

of B-globin

could

ions [M-17]+.

ions in negative-ion

peptides,

and T-13(M=1377)

peptide

all tryptic

ions [M+l]+ and/or

nated molecular observed,

spectra spectrum,

proto-

peptides

T-12a(M=872),

also

were

were hardly

at all. Fig. 1. FD and FAB mass spectra of tryptic peptide mixture of B-globin. Number in parenthe,,ses is the peptide number from the Nterminal end of Bglobin. i

j

:protonated molecular ion (_):dehydrated ion

(,

)

( '):doubly nated

FD mass FD mass

spectrometry

spectrum

was

then applied

of Bs-globin

protonated

molecular

difference

of these

showed

ion of normal two peaks

to the analysis

an abnormal

peptide

corresponded

of abnormal

peak at m/z922

T-l at m/z952.(see directly

protoion

globins.

instead

of the

Fig. 2) The mass

to the difference

of mole-

cular

weights

the abnormal

between peptide

glutamic was

also

acid

and valine.(ref.3)

identified

Doubly

in this mass

protonated

ion of

spectrum.

Peptide T-l of normal B globin (Val-His-Leu-Thr-Pro-Glu-Glu-Lys)

[T-l ]++ .’

m/z

952

CT-,1 ]+

laBa,

Wj_l+’ _

959

900

500

458

Peptide T-l of $ globin (Val-His-Leu-Thr-Pro-x-Glu-Lys) 922

m/z

[T-l]+

missing z

z r

missing ,'

..

:

8,

Fig. 2. FD mass globin(lower).

_

900

5.08

450

1800

.

I

t z 5

spectra

of tryptic

normal

-.

peptides

peptide

1886

958 of normal

M/Z

+globin(upper)

and es-

T-4

_a m/z 1274 (Leu-Leu-Val-Val-Tyr-Pro-Trp-Thr-Gin-Arg)

!-1s.m

abnormal peptide T-4 m/z 1278 (Leu-Leu-Val-Vat-Tyr-m-Trp-Thr-Gln-Arg)

. ,’ : , I ,

Fig.. 3. FD mass

spectrum

(T-13)

of tryptic

peptides

of B-globin

containing

Bx-globin.

212 In FD mass

spectrum

ted at m/z1278

Fig. 3) The mass

: (Pro+Thr) would

or

normal

increment

peptide

would

molecular

However,

proline

T-4,

we concluded

the 36th

position

by only

the latter

is found

peak of peptide

ion of normal

be explained

to aminoethyl-cysteine

since

for proline-at

an additional

the protonated

(Val+Cys).

be changed

Furthermore,

of Bx-globin,

beside

was

only

once

two types

of substitutions

not plausihle,

by the prior Bx-globin

from

the N-terminal

because

cysteine

aminoethylation.

in the amino

that

T-4 was detec-

T-4 at m/z1274.(see

acid

sequence

has a substitution

of

of threonine

end.

DISCUSSION Now we have established small

amount

of sample.

weight

of each

tution

does

not

cause

of peptide.

The

Bx-globin

failed

It is true difficult

peptides happens

We have

demonstrated

the molecular

disorders

these

simple,

ions.

peptide. but this

are suggested chain

(ref.4),

difference "peptide proteins

that

of genetic

conventional

enough of one

primary

peptide

these

very

the substiproperties

methods

have

FAB mass

is competent

been

is rather doubly

ions corroborate spectrum

only when

of tryptic

a substitution

intensity. spectrometry

of genetic

by single several

structures

method

mixture

of coexistent

additional

On the contrary,

and moreover,

mapping"

However,

technique

to be caused

in their mass

when

nor electrophoretic

ions, on account

FD or FAB mass

pathology

and

of the tryptic

molecular

to be in the peptide.with

studying

The

of abnormal

is very

in point

ability

only

the molecular

abnormality.

spectrum

their

method.. It requires us to determine

its unique

in chromatographic

its structural FD mass

mapping"

it permits

exhibits

was a case

ions and dehydrated

the existence

peptide

that

a change

mass

since

the method

in identifying

protonated

little

peptide,

to determine

a "peptide

Moreover,

will

amino

proteins among

facilitate

was

disorders. acid

very

useful

Many

other

substitution

are known various

in genetic

in poly-

to have only

species

the structural

a

and organs: analysis

of

interest.

REFERENCES 1 H. Lehmann and R. G. Huntsman, Man'.s Haemoglobins, North-Holland, Amsterdam, 1974, 478~~. 2 T. Matsuo, H. Matsuda, I. Katakuse, Y. Wada, T. Fujita and A. Hayashi, Biomed. Mass Spectrom., 8 (1981) 25-30. 3 Y. Wada, A. Hayashi, T. Fujita, T. Matsuo, I. Katakuse and H. Matsuda, Biochim. Biophys. Acta, 667 (1981) 233-241. 4 H. Fujii and A. Yoshida, Proc. Natl. Acad. Sci. USA, 77 (1980) 5461-5465.