Mass spectrometry combined with mass spectrometry

International Elsevier

Journal

Scientific

Mass Spectrometry and Ion Physics, 45 (1982)

of

Publishing

MASS SPECTROMETRY F.W.

COMBINED

I.J.

McLAFFERTY,

COHEN,

P.O.

Chemistry

Company,

DANIS,

WITH

AMSTER,

G.H.

Dept.,

Amsterdam

MASS SPECTROMETRY M.A.

BALDWIN,

C.J.

KRUPPA,

Cornell

323

323-329 in The Netherlands

- Printed

M.P.

PROCTOR,

University,

BARBALAS,

and

Ithaca,

F.

M.T.

CHENG,

S.L.

TURECEK

NY 14853

USA

ABSTRACT Tandem mass spectrometry (MS/MS) is a rapidly growing field of high promise for structure determination and quantitative analysis because of its sensitivity, specificity and speed. Rapid progress has been made in instrumentation, including the triple quadrupole and tandem double-focusing instruments, automated with on-line computers. Probably the most valuable application to date is the quantitative analysis of targeted compounds at trace levels with high specificity. However, the extra information available from MS/MS appears to be of particular value for the structure determination of large molecules, such as those best ionized by fast atom bombardment.

INTRODUCTION Tandem

can

individual enhance

the

For

the

sequent

former,

be the

gives

of

laboratories For

those

spectrum

from

MS-I

to

GC/MS

from

its

mass

dissociation

by

MS-I.

This

principal

compounds

of

in

areas

of

mixtures

ions

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Spectrometry"

by

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from

used

of

components

for

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separation

either

which

so

These

other

their

description 1)

sub-

spectrometry.

leading

in

structural

quantitative

a score

these

MS techniques

and

specific

(ref.

of

mass.

ions

its

all

with

conventional

produced

up-to-date

than

as

a characteristic

primary

for

LC/MS, spectrometry)

species by

the

or

and mass

separated

other of

by more

analysis. the

field descrip-

research

field. structure

sample separated

0020-7381/82/0000-0000/$02.75

most

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or

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of

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identification

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then

of

1-4)

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(ref.

secondary

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performance

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of

to peaks

analytical

that

spectrometry

(MS-II)

selector of

mass

determination, adduct)

ion

matrix

in

molecular 0 1982

from fast ions Elsevier

impurities atom then

be or

used

bombardment); provides

Scientific

to

anomolous the structural

Publishing

Company

separate peaks

the (such

secondary information.

324

Alternatively, (EI)

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in then

spectrum

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and

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to

as

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ionization the

separated

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APPLICATIONS

OF MS/MS

Identification

of

Although than

for

components

present

commercial

chromatographic/MS

nesses

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dentification obvious

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to

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by

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changers

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sensitivity

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helpful, for

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10T3

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subpicogram

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instruments

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LC/MS/MS

appear

to

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par-

325 GC/MS/MS An

interesting

case

deacetylmetipranolol, metabolite

of

bolite

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Although

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m/r

Ihe

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266

for

314

to instrument,

267.

higher

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3081266

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Computer

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portant

molecules ions

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biological

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plementary

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ficient

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below

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the 308,

m/z

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metipranolol.

study

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the

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enhancement.

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detection

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6)

'-blocking

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(CH,),-phenyl(OH)OCH2CHOHCH2NHCH(CH3)2,

be an

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value

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masses of

re-

and the

CAD be

elemental

molecular

ion;

326

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weight

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spectra

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the

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technically

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9).

attracted overdose

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System

3-hydroxy

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Retrieval

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these

bold.

327

CH=CH2

3

However, EI

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146

pri-

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for

the

often

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10).

misleading

glycerol

is

be such

ion

spectra little

obtained as

(M + H)+

328

from

adduct

or

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matrix

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peaks, of

Instrumentation. structed 2.3

at

tesla

10

measurement

of

molecules.

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precursor Human

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ther,

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directly

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evidence

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cobalamin

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from

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axial 114

chain peak for

is

spectrum

show shows

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--m/z 1329°~ethylcobalamin

the 1329

132

CAD

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the

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412

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the

2134.

molecule,

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CsI of

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12%,

to

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mass;

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of

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H)+,

Particularly

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spectrum

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ionization

backbone,

1355,

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1.6%.

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sample

of

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abundances of

with

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N-terminus.

for

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""80%

peaks

sequence

sections

up the

of

with

(M t

spectrum

and

peaks

losses

sequence

bradykinins.

are

side

m/z

FAB

yields yields

B-12.

at

posure

as

three

dissociation

represent

centered

from

C-

10,000

possible

2096,

to

eight

the

CAD cross

seriously

the

peaks

1355

and

shows

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Met-Asp-Phe-NH2 from

peak

cm

ions.

IJ/~

intense FAB

spaced

section,

correspond

to

a 60

transmission

produces at

of

up

ion

makes

product

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ions

sequential

CAD cross

less

to

from

the

regularly 8966)

region

these giving

(M + H)+ acids

decrease

metastable

of

the

contrast

that

tripeptide

tive

of

In

angiotensin,

the

MI

K.

for

peptides,

2096

or

of

(m/z

con-

addition

retain

give

showing

mass

that two

FAB

to

spectrometer the

heptadecapeptide

high

latter

to

collectible

the

indicate the

analysis

high

This

in

with

mass

MS-II

yielding

peaks

(M + K)+,

in

mass

+ ions

unusually

information

11).

potential by

Cs35134

(15-Met).

three

(ref.

improved

the

produced

an

ions

gastrin

only

and

shows '

been

allows

of

fragmentation

ions

accelerating

a CAD spectrum

CS35I34

provide

double-focusing

has

which

' ions csn+lln MS-I analysis

15000.

with

12)

KV ion

to these

tandem

(ref.

magnet

using

efficiency. m/z

The

Cornell MS-I

daltons

and

structures

simiof

146

The

final

-CH(CH3)CH2NH-, at

reference

m/z

914. CAD

The spectra;

329 ACKNOWLEDGMENT

Army

We thank

the

Research

Office,

National Durham

Institutes (Grant

of

Health

G29-79-C-0046)

(Grant for

GM-16609)

and

generous

financial

the

support.

REFERENCES

9 10 11 12

F.W. McLafferty (Ed.), Tandem Mass Spectrometry, John Wiley, New York, 1983. R.W. Kondrat and R.G. Cooks, Anal. Chem., 50, (1978) 81A. R.A. Yost and C.J. Enke, Anal. Chem., 51 (1979) 1251A. F.W. McLafferty, Science, 214, (1981) 280. F.W. McLafferty, A. Hirota, and M. P. Barbalas, Org. Mass Spectrom., 15, (1980) 327. W. J. Richter, W. Blum, U.P.Schlunegger, and M. Senn, ref.1, Chap.21. M.P. Cheng, M.P. Barbalas, R.F. Pegues, and F.W. McLafferty, J. Am. Chem. sot. ) accepted. I.K. Mun, R. Venkataraghavan, and F.W. McLafferty, Anal. Chem., 53, (1981) 179. L. Tokes, R.T. LaLonde, and C. Djerassi, J. Org. Chem., 32 (1967) 1020. M. Barber, R.S. Bordoli, G.J. Elliott, D. Sedgwick, and A.N. Tyler, Anal. Chem., 54 (1982) 645A. I.J. Amster, M.A. Baldwin, M.T. Cheng, C.J. Proctor, and F.W.McLafferty, J. Am. Chem. Sot., submitted. F.W. McLafferty, P.J. Todd, D.C. McGilvery, and M.A. Baldwin, J. Am. Chem. sot. ) 102 (1980) 3360.