Determination of active hydrogen content by fast atom bombardment mass spectrometry following hydrogen-deuterium exchange

Vol. 112, No. 1, 1983 April

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

15, 1983

Pages

DETERMINATION

OF ACTIVE

MASS

SPECTROMETRY

Satinder

K.

February

CONTENT

FOLLOWING

Sethi,

Departments University

Received

HYDROGEN

David

of

Medicinal Utah,

of

BY FAST

ATOM

HYDROGEN-DEUTERIUM

L.

Smith

and

Salt

Chemistry Lake

BOMBARDMENT

EXCHANGE

James and

126-131

A.

McCloskey

Biochemistry Utah 84112

City,

25, 1983

Fast atom bombardyent mass spectroyetry following hydrogen - deuterium H20 has been studied as a means of exchanqe in [hydroxyH31glyCWOl and establ;shing active hydrogen content in molecules of unknown structure. one peptide and complex antibiotics, in the mass Nucleotides, carbohydrates, region to 1500 daltons and 29 exchangeable hydrogens were examined, with a correct hydrogen count unambiguously measured in every case. The Imethod is experimentally simple and applicable on a microgram scale, to salts and a variety of polar compounds of biological origin. Knowledge structure This

knowledge

of

an

the

a useful

tization,

or

nuclear

magnetic

methods

become

content

increases.

from

beyond

conditions exchange

Abbreviations

0006-291X/83

is of

4-6 in

atoms

are

principle

FAB, have

useful

hydrogens fast been

atom

groups,

H2

chemical (l),

deriva-

proton -

Unfortunately, the

often

limited

by

active

hydrogen because

back-exchange,

serious

deutethese

favorable

exchange

and

functional

hydrogen

as

but

of by

of

to

under

chemical

errors

bombardment; by

$1.50

126

M, 'H.

molecule

in

of

which

can

ioniresult

(6,7).

exchanged

Copyright 13 1983 bv Academic Press, Inc. All rights of reproduction in any form reserved.

inferred

are

(3-5)

strucand

functional

(2).

Gas-phase

unknown

overall

arrangement

ambiguous

usually

of

following

methods

hydrogens.

non-labile

used: hydrogen

but

be

system

results

spectrometric

sensitivity,

or and

often

of

composition,

liberation

inlet

the

deduction

spectrometry

or

and

Mass

severe

active

source

a molecule

elemental

chemical

mass

the

identity can

by or

inaccurate

microgram-level

zation

directly

of

subunits

the content

resonance,

to

structural

determining

ion

in

complementary

hydrogen

the

content

constraint

of

in

measured

in

hydrogen

important

identities

active

exchange

active

is

role

The

becomes

exact

information

groups.

rium

the

provides

ture.

plays

of

which

all

Vol.

112,

No.

The having

1, 1983

Irecent

development

a major

and

natural

impact

tent

for

molecules

imp-ovemellt

over

spectrome.try

for

been

active

in

of

this

region

The

the in

et

al.

the

have

was

ion were

--Materials. *H20

examined

FAB

not

the

to

were

salt;

several

of

active

would

up from

mass

to

biochemistry thousand

dal-

hydroqen

con-

constitute use

1500

the to

of

a major FAB

mass - *H20

daltons,

and

standpoint

of

a variety

quanosine

spectrum

of

sulfate;

29

applic-

compounds

of

University Utah.

Utah;

A Imixture Blenoxane

glycerol

*H20

of

the

reexchange Mass

at dry

to

rinsed mass

with An

modifications

tip

probe

MSD

of

labeling

and

C-perdeuterioqlycerol,

Isotopes,

St.

uridine

melibiose;

Louis,

MO.

5'-phosphate,

melezitose;

was

The Na

streptomycin

provided

a qift

from

by

W. R.

A*-demethyl,

Dr. and

Dr.

M.

P.

Gray,

B2 were

described

FAB

in

solution

per

Schweizer,

University

purchased

in

2 ul.

*H20.

a 10 ~1

was

adsorbed

pg

rinsed

using

probe

of locally

Prior

Two

syringe

immediately

[hydroxy-*H3!--

~1

that

of

were

application solution

had

transferred

precautions

to the

been to

necessary

pre-

the to

vacuum

avoid

H20.

spectra

magnetic

by

1-5

These by

Tech

prepared

was

tip

The

Mass either

Ion

% *H)

sources:

were

spectrometer.

deuterium

inr,trument,

atom

a concentration

*D20.

spectrometry.

potential.

A2,

probe

the

with

of

(Y7.8 from

was

Samples

the

transferred

vic~usly

extent

a fragment

Laboratories).

(1:l)

sample,

of

labeling

N-[(9-6-D-ribofuranosylpurin-5-yl)-

MI

bleomycins

preparation. -

the

of (Bristol

Sample

METHODS

Na salt

conotoxin

formation

The

AND

y-cyclodextrin. 5'-phosphate,

of

for

on.

Na salt;

B-cyclodextrin;

glycerol of

(11).

commercial

5'-phosphate,

deuterated

mechanism

purchased

from

of

the

commented

purchased

carbamoyllthreonine

tht!

to

the

accuracy,

use

study

[Hydroxy-*H3]glycero1 ~ atom % *H) were

(9S+

followinq

lotk

in

is

[hydroxy-*H3]glycerol

region

high

employed TpT,

negative

matters

were

problems up

in

weight

with

spectrometry

and

exchanged

molecular

mass

a consequence,

MATERI4LS

of

region

attractive, As

technique

deoxynucleotide

related

as

structural

COMMUNICATIONS

importance. Sindona

and

bombardment

mass

is

directly

the

RESEARCH

determination

methods.

materials in

the

accurate

a microgram-scale,

0'1

ion

solution

of

existing

hytjrogens.

biological

of

atom

in

prospect

{explored

ability

the

BIOPHYSICAL

fast

chemistry

The

(B-10).

AND

of

on

products

ton5

has

BIOCHEMICAL

were or

electric

11N

ion

source

by

Biemann

and 127

using

dcquired

scanning was

a Varian at

installed,

co-workers

8 kV

731

accelerating

closely (12).

MAT

folloriny

A neutral

Xe beam

Vol.

112,

having data

No.

1, 1983

6 KeV

energy

were

Nicolet

taken

was

from

1170

Ion

BIOCHEMICAL

the

to

data

intense the

(Mt'H-1)'

replaced

by

protium.

teristic

of

FAB

for

1.

In

those In

from

Table

RESEARCH

desorption.

In

accumulated

RESULTS

AND

the

FAB

mass

are

given

in

within

each

exchanged

molecule

notation

Table

from

peak

fully

protonated

ion

spectra

exchange

[most

refers

for

BIOPHYSICAL

over

some

COMMUNICATIONS

cases

ion

(1024

scans)

2 min.

abundance using

a

averager.

abundance

the

mass

siqnal

hydroqen-deuterium to

used

AND

ion

Additional

every

(17,18),

case

the

of

Table

isotopic

compounds

Abundances

cluster.

in

a deuteron,

one

such

as

Nat

adducts,

were

also

observed,

but

extent

of

exchanqe

in

b,y

analoqy

FAB

to

(16).

has

which

these

(M+2H)+

in

deuterium

ions

normalized

notation

produced

which

following

are

The

to

usually

species

ten

1.

attached

(unexchanqed) to

spectra

spectra

molecule,

MHt

refers

DISCUSSION

The

been

are

charac-

are

not

listed

ions

was

the

in same

as

reported. every

the

case

mass

1.

examined

shift

Sull~nlary

the

between

of

the

Molecular

active

spectrum

hydroqen

of

Weight-Related

Molecular Weight., Unlabeled

Compound

correct

the

Ions

Number of Active Hydrogen8

Percent Exchange

content

unlabeled

was

material

Following

established (data

Hydrogen-Deuterium

nl/z

(relative

not

Exchange

intensity)"

(M+'H-3)+

(M+~H-~)+

(M+'H-l)+

(M+?H)+

90

345(O)

350(19)

351(55)

352(100)

512

390(13)

391(26)

392(60)

393(100)

514

93

520(11)

521(1Y)

522(56)

523(100)

Melibiose

342

90

349jiO)

350(41)

351(94)

352(100)

Melezitose!

504

53

514(13)

515(34)

516(80)

517(100)

581

93

59607)

597(59)

59X(100)

599(51)

u-Cyclodextrin

972

Y3

989145)

990(X3)

991(100)

y-Cyclodext&

1296

93

1319(66)

1320(87)

1321(100)

132L(3uj

Bleolilycins~ AL I cation< dwethyl-AZ G:

1414 1399 1424

93 93 93

1436(83) 1423(71) 1452(87)

1437(95) 1424(88) 1453(9Y)

1438(100) 1425(100) 1454(100)

1439(9X) 14X(96) 1455(90)

Conotaxin

1492

92

1518(79)

1519(95)

1520(100)

1521(x')

Uridlne-5'.phosphate, nlonosodlum salt

346

Guanosine-5'-phosphate,

385

id-[(9-6-U-ribofuranosylpurine 6-yl)carbomyl]threonine phosphate, ,monosodium

Streptomycin

sulfate

MIC

6

5'. salt

27

d Each isotopic cluster is normalized to the largest peak in the cluster. p FAB amass spectrum of unlabeled compound, ref. 13. d FA6 mass specttul of unlabeled cwlpound, ref. 14. Ion m/z 1439 IS fully exchanged M+; lower amass peaks dlie to bath exchange. c Tetradecapeptide, witn 2 disulfide bonds. ref. 15.

128

Y92(&5)

Vol

112, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(Mt2H)+ 517

m/z Fig.

1.

Fast 2

atom

bombardment

H3]glycerol

(G)

mass

and

spectrum

'H20.

The

of

m/z

meleritose

517

ion

in

[bdroxy-

contains

12 deuterium

atoms.

shown),

and

derlce thl?

was

extent

can

visual

required

be

if

the

the

established

as

The

observed for

29

the

hydroqens.

glycerol

judged

No evi-

skeleton

from

spectra

ranged

frolrl

from

illustrated

by

intensity

into deter-

heavy

simply

can

the

unambiguously

the

spectrum

be is

mass

129

518

spectrum

and

is

fewer

than

the

18

2Hlll-

accounted

can of

into

[hydroxy-

mainly

or

the

established of

calculated

known

as

of

introduction

lUO0

isotopes,

composition from

before m/z

m/z

90-Y%

decrease

was

of

contributions

elemental

(below

content

natural

steadily

times

cases

deuterium

relative

to

longer

simpler

as

isotope the

from

ions,

was

stand

inspection,

heavy (19)

to In

contribution

natural

of

[hydroxy-'H3]glycero1

value

allowed

1).

hydrogen

fragment

frown

This

was

(Fig.

also

any

exchange

h,ydrogens),

melezitose

equations

of

a maximum

matrix.

spectrometer.

simple

tice,

or

possible.

exchangeable

the

incorporation

of

sample mass

up to

C-perdeuterioglycerol

maximum

la.)eled

by

for

exchanqe,

tnolecule

us*ng The

th?

following

found

protonated

~mined

th?

that

13C .

for In

by

standard

be

estimated;

unlabled

by prac-

it coin-

Vol.

112,

pound.

No.

1, 1983

The

lower

incomplete

from

deuterium

peaks

are

Recause at

the

time

of

glycerol

ions

exchange

of

sample number

of and

572

the

(*HI,)

(20) from

If,

functional

the

isotopic

be

qained

by

lower

isotope

in

ohtdi

ned.

ble,

particularly

glycerol

(Kf

or

Na+)

micrograms

of

sample

cases

Reduction

conventional

in

1.

Fig.

21%

pattern

of

very

may

appear

pattern,

sample to

accu-

whether

levels

of and

other.

The

comparison

of

ions

(e.g.,

visually

effect m/z

517

the

confusing.

heavy

usinq

used

= 3,4,5...)

large

natural

the

of

each by

For

for

peak

(n

illustrated

an of

for ions from

isotopes,

deuterium

intense

and

by

levels

averager scanninq

was

size

in

of

several

to

their

suspected, to

the

sodium from

acidic

acidification establish

or

this

present

study

minutes

duration

submicrogram over Some

(Fiq.

tormation

used

weights

used

salt

(12,lH)

signal

scan.

as

fact

can

tip.

minimized. to

examined

were

were

molecular

scan

were

structure,

probe

sample

is

1)

experilnents

the

a signal

is

of

regardless

ions

having

(20).

be

the

cases

isotope

content

Thus,

be within

ions

lower

can

to

some

content

clusters sample,

mainly

in

second

deuterium

interest.

pattern

due

For

deuterium

the

carboxylate)

on

most

to

are

possible

and

the

of

be

first

isotopic

unknown

directly

encountered

by

of

(phosphate,

cluster

content,

overall

(Table

a molecule

out

In

in

correcting

mononucleotides

Several ience.

the

in

1)

ions.

replacement

carried

the

l),

the

may

cluster

clusters

groups

cation

region

found

glycerol

three

salts.

mass

Fig.

COMMUNICATIONS

material).

of

glycerol exchange

on

Mt

represent

of

typically

can

The

the

RESEARCH

in

unlabeled

changes ions

extent

Table

measured

of

abundances

matrix

515

from

spectrum

small

deuteriums

calculating

taken

to

were

Clarification

&as

the

appear

515,

([hydroxy-2H31glycerol)n.zHt

bleom,ycins,

be

atoms,

the

ions,

6$*)

mass

ionization,

measure

(m/z

BIOPHYSICAL

contributions

the

glycerol

AND

peaks

but

sensitive the

rately

of

isotope

exchange,

(recognized many

BIOCHEMICAL

For

n/z

cases,

will

also

I30

in

although suffice.

is

where

ITIeaSlJrWWflt

some I)

600,

fluctuation

accurate

in

levels

ion

for

convenwas

therefore

possi-

interference

from

beam of multiple

intensity deuterium scans

Vol. 112, No. 1, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS This work was supported by the National Institute of General Medical Sciences (GM 21584). The authors are grateful to Dr. W. R. Gray for advice and assistance in calculation of isotopic abundances, and to Mr. S. A. Martin in the laboratory of Dr. K. Biemann for advice concerning ion source installation. REFERENCES 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Ma, -r. s.,

and Ladas, A. S. (1976) Organic Functional Group Analysis by Gas Chromatography, pp. 138-145, Academic Press, New York. Budzikiewicz, H., Djerassi, C., and Williams, D. H. (1964) Structure Elucidation of Natural Products by Mass Spectrometry, Vol. 1, pp. 17-40, Holden-Day, San Francisco. Hunt, D. F., McEwen, C. N., and Upham, R. A. (1972) Anal. Chem. 44, 12921294., Blum, W., Schlumpf, E., Liehr, J. G., and Richter, W. J. (1976) Tetrahedron Lett. 565-568. and Smith, L. L. (1979) Biomed. Mass Spectrom. 6, 15-18. Lin, Y. Y., and Sethi, S. K. (1980) J. Am. Chem. Sot. 102, 6953-6963. Hunt, D. F., Martinson, 0. P., and Buttrill, Jr., S. E. (1976) Org. Mass Spectrom. 11, 762-772. Rinehart, Jr., K. L. (1982) Science 218, 254-260. Dell, A., and Morris, H. R. 11982)Biochem. Biophys. Res. Commun. 106, 1456-1462. Barber, M., Bordoli, R. S., Sedgwick, R. D., Tyler, A. N., and Green, B. N. (1982) J. C. S. Res. Commun. 936-938. Sindona, G., Uccella, N., and Weclawek, K. (1982) J. Chem. Res. 184-185. Martin, S. A., Costello, C. E., and Biemann, K. (1982) Anal. Chem. 54, 2362-2368. Kambara, H., Ogawa, Y., Mochizuki, K., and Hishida, S. (1982) Mass Spectrosc. (Tokyo) 30, 169-181. Barber, M., Bordoli, R. S., Sedgwick, R. D., and Tyler, A. N. (1981) Biochem. Biophys. Res. Commun. 101, 632-638. McIntosh, M., Cruz, 1. J., Hunkapiller, M. W., Gray, W. R., and Olivera, B. M. (1982) Arch. Biochem. Biophys. 218, 329-334. Barber, M., Bordoli, R. S., Sedgwick, R. D., and Tyler, A. N. (1981) 293, 270-275. --Nature Bordoli, R. S., Garner, G. V., Gordon, D. B., Sedgwick, R. Barber, M., D ., Tetler, L. W., and Tyler, A. N. (1981) Biochem. J. 197, 401-404. Barb'er, M., Bordoli, R. S., Elliott, G. J., Sedgwick, R. D., and Tyler, A. N. (1982) Anal. Chem. 54, 645A-657A. Beynon, J. H. (1960) Mass Spectrometry and its Applications to Organic Chemistry, pp. 294-302, Elsevier, Amsterdam. and McCloskey, J. A. (1977) Anal. Chem. 49, 281-283. Yamamoto, H.,

131