Mass spectrometry of polar complex lipids analysis of a sulfatide derivative

BIOCHEMICAL

Vol. 37, No. 1,1969

MASS

AND BIOPHYSICAL

SPECTROMETRY

OF

ANALYSIS Karl-Anders

OF

A

Karlsson,

Institute

of

COMPLEX

SULFATIDE

Bo

Medical

POLAR

E.

RESEARCH COMMUNICATIONS

LIPIDS

DERIVATIVE

Samuelsson

Biochemistry,

and

Fack

400

Goran 33,

0.

Steen

Gbteborg

33,

Sweden.

Received

August

1,

1969

Summary. Acetylation of brain sulfatide followed by trimethylsilylation at elevated temperature produces a fully acetylated derivative with the sulfate group replaced by a trimethylsilyl substituent. The mass spectrum of this compound is dominated by peaks from the galactose residue, and a 3-position of the original sulfate group may be assigned.

Tissues kidney,

with the

have

rectal

high

The

ratio potassium

of

the

same

the

be

rectal

cat

(3)

examination

of

subcellular

for

the

role

of of

and

metry

is

a method

of

and

allows

analysis

Mass

spectra were

investigation with after of

on

silylation, hydroxy

sulf

the

as

(1).

of

these

systems

of

has

(4),

was

been by

In Attempts

22

also

to

in

spectroother

elevated

lipid

material.

glycosphingoanalysis

present

of work

analyze

a silyan

this

lipid

diazomethane

However, at

in

necessary

of

with

unsuccessful.

cells

a closer

Mass

to

treatment

silylation

was

neutral

the

hormone

blood

and

successful

(5).

red

powerful

of

the

For

amounts

and

after

far

it

indi-

enzyme

analysis.

proved

made.

may in

of

weight).

gland,

reported

This

sodium-ion

content

salt

sodium approximately

sulfatide-

g dry

method it

the is

receptor the

submicrogram

portion, so

gland,

ester)

(1).

or

derivatives

been

followed

salt

acid

self-supporting

in

avian

of

atide

have groups

gland

sulfatide

sulfatide

choice

sulfate

only

(1 p. g per

presented has

a retained

with

trimethylsilyl

recently

ganglioside

salt

brain,

avian

activity

Assuming

sensitive

fields

lipids

the

a carrier

the

low

a specific

the

(2)

the

as

calculate very

of

and

cells

be

fractions

develop

and

transport.

may to

and

like

(cerebroside-sulfuric

involved

same

one

the

elasmobranchs

gland

is

the

transport,

adenosinetriphosphatase

sodium-ion

transport,

lated

of

concentration

sulfatide

to

sodium-ion

sulfatide

sulfatide

for

that

of

of

dependent

dependent ratio

increased

gland

levels

and

cate

an

temperature

acetylation

or

BIOCHEMICAL AND BIOPHYSICAL

Vol. 37, No. 1, 1969

results

in

oxy

group,

an

indication

a quantitative the

exchange

product of

the

of

and

by

column

analysis

sphingosine composition duced

in

the

the

sole

and

a few

is

heterogenous

and

.at

room

screw

The

les

sucked

with

for

1 hour

at

was

a water

pump

120’

C with

the

residue

taken

For

probe

a quantitative

quartz

tube

spectra

may

were

with ionizing

inlet

below

cerebroside

and

to

40

and

sulfatide

peaks

second

at

corresponding

the

30-unit

acetoxy

groups

for

and

this

of the

of mass

galactose

molecular

peaks

above

The

peaks

are

those

major at

m/e

264,

169

was

mg

treated

evaporated a

sample

sample, is

per

ml).

however,

a

evaporated.

resolution

source

Mass

instrument of

70

eV,

temperature

of in (10)

of

of

are

sulfatide

1 and and

be m/e

220°C.

is (see

an

exchange

to

m/e

900,

the

exist

fatty

two the in

23

spectra carbohydrate both

respecti-

used

methy-

for

the

due

at

to

331, in

is

at

of

one

spectra.

acid look

interacetyl

m/e

formula

due

Fig.

m/e

1).

361,

of

group.

a heterogenous

from

2,

silylated

partially

43,

derivative to

and

a trimethylsilyloxy

(due

109

Fig.

cerebroside

owing

derived

at

oxygen

the

acetylated

may

cases

for

and

samp-

were

potential

reproduced

peak

in

series

high

spectra

glycosidic

peak increase

902

acetylated

both

the

of solvent

carbohydrates in

the

reproduced.

fully

largest

a fragmentation

The

on

a teflon-faced and

spectrometry

(10

the

ion

respec-

samples

mass

ionizing

an

Mass

(11)

Base The

not

are

data

MS an

with

reagent

amounts

AEI

and

were

acetylated

pretation. ions.

an

of

pyridine-hexamethyldisilazane-

solvent

which

mA,

discussion.

Both

reprocent

l:l,

nitrogen

For

small

using

0.1

and

Published

lated

from

of m/e

Results

vely.

used, on

tube

The

the

acid

species,

a test

with

fatty

per

anhydride-pyridine

(9).

into

20

sulfatide

hour.

were

species

about

acetic

chloroform.

system,

current

Peaks

in

of

recorded

a direct

an

con-

bases

molecular up

methods

lipids

long-chain

under

caps

were the

The

the

in

v/v/v

transfer be

of with

for

dipped

Both

the

the

evaporated

1,

up

was

(1,6).

2 makes

cent

closed

4:2:

pyrophylite

and

overnight,

reagent

trimethylchlorosilane and

salt)

, and

performed

temperature

cap.

(lithium procedure

1 and

15 per

bovine

This

dihydrosphingosine.

(7,8)

with

pure

, and of

Fig.

was

spectrum

position.

elsewhere

cent

about

Acetylation

v/v,

per

of

a trimethylsilyl-

sulfatide

carbohydrate

formulas

cerebroside

tively.

brain

presented

as

for mass

chromatographically

human

been

galactose

sulfate

group

chromatography.

have

tained

sulfate

Thin-layer

cerebroside

obtained

the

a characteristic

original

Experimental. brain

of

giving

RESEARCH COMMUNICATIONS

the Except

portion) very

similar.

residues, The

of

and first

of

BIOCHEMICAL

Vol. 37, No. 1, 1969

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I , 331

M=1079 718 I 1

; I I l

I I CH3 Wl3l~3-CH=CH-~H

0 lo iH3

$tl -

CH2 +O I

NH

; 0 I

OCOCH3 COCH3 0 0

OCOCH3 CH2OCOCH3

do 4OCOCH3 &H3)2,

100.

hH3

J

Fig. 1. Mass spectrum of an acetylated mixture of bovine brain cerebrosides. in the top formula contains galactose, carbon 2-hydroxy fatty acid.

24

and trimethylsilylated The molecular sphingosine and

species a 24-

BlOCHEMKAL

Vol. 37, No. 1, 1969

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I , 361

MS 1109

CH3 KH$,Z-CH=CH-~H-~H-CH~, ? co :H3

NH

I

CH20COCH3

I

do H;OCOC”3 t;H*l3, ;H3

im-

40. 20. 5”

Fig. 2. Mass spectrum mixture of human brain the top formula contains in 3-position), sphingosine

of an acetylated The sulfatides. galactose (with and a 24-carbon

25

and trimethylsilylated molecular species an original sulfate 2-hydroxy fatty

in group acid.

BIOCHEMICAL

Vol. 37, No. 1, 1969

these

has

not

elemental

yet

from

60-42

(60

mass

( and

of

thus

otherwise

the low

suggests

a pyranose

therefore

of

ether

to

3-methoxy

The

peak

at

2-,

m/e

Fig.

169.

2 is

by

in

3-position,

The

of

peaks

bond

at

by

use

of

the

C-l

by The

layer

addition

derivatives) The

of

information methods.

human

brain

tose

residue

(m/e

43)

of In

the

case

peak

at

(compare intensity.

of m/e

Fig. In

trimethylsilyl

(14)

of

results

obtained the

this

nature

way,

may

be

spectrometry. with

of

e. g.

sulfatide

determining

the of

phosphate)

is

not

thindetection.

the

sulfate or

polar

polar

diglycosyl

of

easily

encouragroups

lipids with

in

present-

diglycosylceramide from 30-40s

derivatives

26

often is

polar

complex

obtained

derived got

portion, lipids,

of

acetylated

has

glycosidic

(monoglycosyl

from

or

33 1, l),

in

characterization

fully

group.

compared.

for

which

the

of

means

derived

carbohydrate

sulfate

flame

chromatography

possibilities

e. g.

general,

peaks

after

a trimethylsilyloxy

the

no spectrum

with

combination

(15)

are

the

original

obtained

sulfatides

origins

homogenous

(containing

day

different

suggests

way when

referlook

concerning

sensitive

a simplified

predominance

and

a very

e. g.

or in

peaks

that

a confirmation

used

or

give

indicate

not

On

compounds

Information

be

(11).

the

probable

(718-60)

is

methoxy

isomers

the

are

(acetoxy

with

acetylated

(13)

may

useful of

obtained ging

offers

658

the

acetates

very

2

possibilities.

3-position

compound

absorption

as

to

in

Fig. spectra

spectra

comparable

positional

(sulfate),

atomic

reference these

replaced

This

method

it

4-,

(12).

substituent

position,

fully

the

m/e

methods

e. g.

the

in the

substituent

between

comparison

a pyranose

sphingosine.

present

in

having

chromatography

group

is

appears

around

classical

replaced

gained

In

of

of

therefore

produced

substituent series

and probably

It

No

an

excluded,

199 of

sulfate

substituent

and

be

to

hexapyranose the

by

changed

4.

331-60-

obtained

thus

original

or

be

peak

trimethylsilyloxy

appearance

the

the the

which

is the

has

ketene),

may

have

group

lost,

and

109

general

2,3

169

of

of

conclusively silyl

that

6.

should

these,

the

above

similar.

42

m/e

group)

monomethylated

easily

to

and

(IO),

bound

most

suggested

6-position

the

position

of

is

169 As

in

with

sulfate

differentiate

isomeric,

methoxy)

in

be

the

spectrometry

m/e

identical

position A

structure

to

mass

red

at

should

group

original

amounts).

available

However,

the

peak

in

in

acid.

at

thus (lo),

acid

group

acetic of

(present

acetic

RESEARCH COMMUNICATIONS

peak is

hexoses

of

acetoxy loss

group

acetylated

the

a retained

The and

C8H904,

fully

is

additional

yet

identified.

composition

derived

has

been

AND BIOPHYSICAL

the of

of

terminal the

(4,5),

base

galacpeak galactosyl-

BIOCHEMICAL

Vol. 37, No. 1, 1969

ceramide base

of bovine

peak,

manner 90).

intensity)

trimethylsilyl

ry , peaks

kidney)

has

strated

study

like

suitable

these

of natural

derivatives

(50-600/O

peak

at m/e

hexosamine

of

in an analogous

trimethylsilanol

(451-

of tetraglycosylceramide 420,

(globowhich

is the

On the contra-

derivative. lipophilic

portion

are

of

1 and 2).

analysis will

peak

derived

lost

the heterogenous, by mass

definitely

products.

systematic must

361,

an intense

Fig.

direct

in detail,

and synthetic)

from

(compare

A successful of compounds

at m/e

largest

1 and having

N-acetyl

obtained

intensity

second

derivative

of the terminal

in the

has the

331 in Fig.

of human

mass low

73,

to m/e The

side

m/e

brain

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

spectrometry

broaden

However, works

be performed,

on known including

of mixtures

the before lipid

use

of this

this

can be demon-

structures

a search

for

method (natural

the most

of analysis.

Acknowledgement. The present work has been supported by grants from The Tri-Centennial Fund of the Bank of Sweden and The Swedish Natural Science Research Council (Project 2077-34).

References. 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Karlsson, K. -A., B. E. Samuelsson and G. 0. Steen, Biochim. Biophys. Acta 176, 429 (1969). Albers, R. W., Ann. Rev. Biochem. 36, 727 (1967). Bonting, S. L., K. A. Simon and N. M. Hawkins, Arch. Biochem. Biaphys. 95, 416 (1961). Karlsson, K. -A., To be printed in Proceedings from a Symposium on Chemistry and Metabolism of Sphingolipids, held at Michigan State University, East Lansing, U. S.A. , on May 5 and 6, 1969. Sweeley, C. C., Personal communication. A paper has been submitted to this journal. Karlsson, K. -A., B. E. Samuelsson and G. 0. Steen, Acta Chem. Stand. 22, 2723 (1968). O’Brien, J.S., and G. Rouser, J. Lipid Res. 2, 339 (1964). Svenne rholm, L. , and S. StXllberg-Stenhagen, J. Lipid. Res. 2, 215 (1968). Hashizume, T., and Y. Sasaki, Anal. Biochem. E, 199 (1966). Biemann, K., D.G. DeJongh and H.K. Schnoes, J. Am. Chem. Sot. 85, 1763 (1963). DeJongh, D. C., and K. Biemann, J. Am. Chem. Sot. 85, 2289 (1963). Stoffyn, P. , and A. Stoffyn, Biochim, Biophys. Acta 2, 218 (1963). Roe, D.A., P.S. Miller and L. Lutwak, Anal. Biochem. E, 313 (1966). Gersude, K., Anal. Biochem. 25, 459 (1968). Stoffyn, A., P. Stoffyn and E. MHrtensson, Biochim. Biophys. Acta 152, 353 (1968). 27