Pyrolysis mass spectrometric studies on mycobacteria

Interrtafional

PYROLYSIS

J.

Publishing Company,

MASS

SPECTROMETRIC

HAVERKAMPl,

G. WIETENl

Amsterdam

STUDIES

and D-G.

Institute

of Public

- Printed in The Netherlands

ON MYCOBACTERIA

GROOTHUIS*

lFOM-Institute for Atomic and Molecular Amsterdam (The Netherlands) 2National

67

and Ion Physics, 47 (1983) 67--70

Journal of Mass Spectrometry

Elsevier Scientific

Health

Physics,

(RIV),

Kruislaan

Bilthoven

407,

1098 SJ

(The Netherlands)

ABSTRACT Application of Curie point pyrolysis-quadrupole mass spectrometry together with pattern recognition methods for spectrum evaluation of-Fe-;-s the possibility for rapid characterization of complete bacterial cells. The spectral fingerprints obtained from mycobacteria are used for identification of Tuberculosis complex strains and for quality control of M.leptrae prepared from .ivzwiuo cultures. Pyrolysis-collisionally activated dissociation-MS can be used for structure elucidation of specific pyrolysis fragments. INTRODUCTION Complex

and macromolecular

dily be characterized thermally mixture

fragmented of largely

characteristic Curie

point

riety

of

(usually

in use in routine

Some

data

MATERIALS

obtained

isolated

specific) and

samples.

microbiological laboratories.

of the spectral

to common

practice

the field

Py-MS

procedures

on unweighted,

that

va-

are normally of

classification will

studies.

Py-MS

by computer

weighted,

of mycobacteriology

can be

for a great

advantage

for evaluation

is routine

classification,

substituting

A special

a

profiling

testing,

in conventional

can be based

within

data

on our

and immunological

tool, test

of which

chemical

viz. heterogeneity

are

yielding

projects

microbiology,

In

epidemiological

as an all-round

research

integral

can rea-

is

techni-

and identi-

or "key"

features.

be described

below.

AND METHODS

M.tube&c&u&&, were

may be used

analysis

applications

strains,

the composition

One of the main is the

cells

Molecules

(Py-MS).

in the MS-vacuum

molecules,

1).

system

of purposes,

accessibility

In analogy

fication,

EIMS

of unknown

the method

ques.

fragment (ref.

like bacterial

spectrometry conditions

of microbiological

for a variety

Thus,

materials

controlled

volatile

Py-quadrupole

identification

the easy

under

for the sample

("fingerprinting") applied

organic

by pyrolysis-mass

from

iM.buv-in,

M.buvti

from our

RIV culture

armadillo

liver were

of the UNDP/World

Bank/WHO

OOZO-7381/83/0000-0000/$03.00

through

0

BCG and other collection obtained

("atypical")

(ref. 2). from

Dr. P. Draper,

1983 Elsevier Scientific

Samples

the IMMLEP

mycobacteria of M.Rephae.

Steering

Natl.Inst.Med.Res., Publishing Company

Committee Mill

Hill,

London.

Samples

phosphate

were

buffer

quadruplicate.

without

are: Pyrolysis

any further

time

16-170;

see

carrier rate

pretreatment.

ref.

Fe/Ni

1 s; MS-inlet scan

out of a suspension

of the Py-MS

data analysis sample

heating

range m/z

onto the Py-wires

For a description

for multivariate total

applied

Each strain

system

(Curie

point

15O'C;

10 scans/s;

instrumental

51O'C);

temp.

EI electron

total

or

analyzed

in

and the computer

1-4. Typical

temp.

was

in water

scanning

conditions

rise time 0.1 s;

energy

time

methods

14 eV; mass

15 s (signal

averag-

ing).

IDENTIFICATION

OF CLINICALLY

Identification

RELEVANT

of mycobacterial

10s is compl ex (M .tub~ticuLuhih species

can

selected were

readily

"key" mass

a high

ratio

and long-term

stability

spectra

is derived

from

Extensive 59,

selection

cal series

order

of the spectral ference

to assign

a peak

ions which TBC

among which

cell wall

also

enable

complex found

set of empirically

The selection

the

criteria variance groups

reduced

TBC-complex

(6 peak)

spectrum

which

strains. set of key peaks:

strains

analyzed

identified

to conventional

m/z 50,

by this

proce-.

as atypical)

strain

typing).

and

A typi-

1 in the form of a non-linear space

for a set of unknown

like

elucidation the main

components cytosols observed

but

map

and re-

components

strains

to identify origin

as expressed

C2H-J);) interpreted It should

arabino-galactan. components

spectra. mainly

The

in the cell-wall-derived

as derived be noted

spectra

Py-CADMS

spectra.

spectra.

different

of cell wall

carbohydrate

Recent-

of the differences

can contribute

Py-CADMS

the neutral

cell.

contributing

by the key mass

a Py-fragment

each

it is difficult

of the bacterial

key mass m/z 59 represents

also cytosol

in whole-cell

that in general

Consequently,

of the molecular

(probably

represent

fragments.

(most likely

cell wall

is such

(ref. 5) of key peaks

and atypical that

of bacteria

chemical

M+' of acetamide

ty of key masses was

"atypical"

TBC and atypical

in a best

in Fig.

spectra

study

and an EI-fragment

carbohydrates

bacterial

mean

(i.e. a TBC strain

(pyrolysis)

to specific

a Py-CADMS

might

it was

glycan)

spectra.

between

200 mycobacterial

in the 6-feature

of Py-mass

up of several

ly we started

Thus,

or to other

variance/intra-strain

reference

(all relative

the tubercu-

strains.

is made

between

BCG)

used to compare

resulted

is represented

differences

The complexity peak

selected

errors

errors

of analyses

was

a hypothetical

studies

116. About

gave 0% first-order

7.6% second

with

a set of nine

peak

inter-strain

of variance

to either

of a small

Py-mass

of the discrimination

strains

71, 81, 98 and

dure

intensity

analysis

of unknown

as belonging

out on the basis

in the complex

of peak

(ref. 2). One way

strains

M.bovd and M.bov&

,

be carried peaks

MYCOBACTERIA

ions peptidofrom neutral

that

not only

to the intensi-

of m/z 59 of mycoEI-fragment

that

69

6 key features-

m/z

Non

50,59,71,8i,96,116

Linear

Map

:5.2)

(stress

Fig. I. Non-linear map of Py-mass spectra of a set of mycobacterial strains. Replicate spectral points are connected; distances between points represent spectral differences. Shaded strains are M.~:ub~c~u~~ D , M.bovi~ •lland M.buu& BCG q references. Unnamed strains belong to the TBC complex, named strains represent atypicals and are remote from the TBC complex. The M.uvim strain (bottom right) was correctly typed as atypical.

CHARACTERIZATION M.Pephac of infected and includes (toxic)

OF MYCOBACTERIUM

LEPRAE

can only be cultured

in uiwu

armadillos_ enzyme

chemicals

the final

on the basis

have

about medical

of traces

A-G which

and washing

procedure

MES and Percoll

the presence

peaks,

possible

are final

due to omission TWEEN

In

products samples

contaminants

studies

be detected.

This

compound

is

by Py-MS

absent

Fig. 2 shows

in the

a plot

the PEG concentration.

purification

respectively,

in

of M.Xcptiac.

procedure

J and K, and H and I, PEG levels

or inefficacy, C&I

undesired

can be introduced

89 (almost

components).

tissues

is complicated

be performed

e.g. m/z

of an extensive

from

by which

of such

and immunological

of PEG can rapidly

fragment

in bulk

steps

of m/z 89 in M.&epfiae preparationsvetiita

very low PEG levels. Analogously,

obtained

and purification

HEPES,

of pure M.Cept~ae and other

ceptable, step.

Knowledge

of characteristic

of the intensity Samples

PEG, TWEEN,

detection

and was

extraction

in bacteriological,

Semiquantitative

spectrum

like

preparations.

prerequisite

isolation

The

treatment,

PREPARATIONS

and

are unac-

of the final washing is structurally

related

70

l.O-

O.B-

0.6 i

L

ABCDEFGHIJK M. leprae BATCHES

2b 4b 6b Concentr. PEG (ppm)

Fig. 2. Concentration of PEG in M.Lcp&ae preparations; l represent standard contaminated with PEG), o represamples (from one M.&qzzae batch, deliberately sent M.Rcpaae batches to be tested.

to PEG but

can be differentiated

sib 1 e Percoll ker peaks

and MES contaminations

in the Py-mass

can also be applied pur fied M.&~&W cat

ve of changes

M.Rep~e

by specific

spectra.

in the study

preparations.

signals

can also be detected

Apart

from

of biological

detection

complex

contributions

were

units.

on the basis

of these

heterogeneity

In view of this,

in cell wall/cytosoT

of its polyol

Pos-

of mar-

artifacts

Py-MS

of extensively

dissimilarities observed

among

indisome

preparations.

REFERENCES 1 2 3 4 5

H.L.C. Meuzelaar, J. Haverkamp and F.D. Hileman (Eds.), Pyrolysis Mass Spectrometry of Recent and Fossil Biomaterials; Compendium and Atlas, Elsevier, Amsterdam 1982. G. Wieten, J, Haverkamp, H.W.B. Engel and L.G. Berwald, Rev.Infect.Diseases, 2 (1981) 871-877. J. Haverkamp and P.G. Kistemaker, this Volume. L.G. Berwald, D.G. Groothuis and P. Draper, Ann. G. Wieten, J. Haverkamp, Microbial. 1338 (1982) 15-27. G.J. Louter, P.F.M. Stalmeier, A.J.H. Boerboom, J. Haverkamp and J. Kistemaker, Z.Naturforsch. 35C (1980) 6-11.