Mass Spectrometry of Biological

40 Pyrolysis/Gas Chromatography/Mass Spectrometry of Biological Macromolecules E. REINER and T. F. MORAN Downloaded by UNIV OF TENNESSEE KNOXVILLE on November 14, 2016 | http://pubs.acs.org Publication Date: June 1, 1983 | doi: 10.1021/ba-1983-0203.ch040

1

Georgia Institute of Technology, School of Chemistry, Atlanta, GA 30332 In pyrolysis/gas chromatographic studies of biological macromolecules, more definitive information is attained by adding a mass spectrometer and data system to the combined instrument. Key peaks that impart specificity or uniqueness to the pyrogram (fingerprints) can be identified by mass spectrometry. Consequently, microbial or mammalian cells can be characterized with greater confidence through their pyrolysis fragments. Highlights of studies on pathogenic microorganisms and mammalian cells are discussed. The latter include primary normal human tissue cells, cultured cells reflecting genetic disease, e.g., cystic fibrosis, and cells involved with malignant disease processes. Also included is a discussion of the possible use of computers in disease diagnosis.

FROM

OUR INITIAL WORK w i t h pyrolysis/gas chromatography ( P y / G C ) techniques i n 1964, w e suspected that the small molecular w e i g h t series of peaks that comprise the pyrogram were u n d o u b t e d l y fragments split off from macromolecular structures. Several lines of e v i dence pointed i n this direction. T h e peaks that p r o v e d to be u n i q u e or specific for a strain of pathogenic microorganisms were also closely associated w i t h their antigenic structure. (See T a b l e I.) C o n s i d e r a b l e antigenic activity resides i n c e l l w a l l biopolymers, and the association has b e e n confirmed by numerous structural studies on the chemistry of c e l l walls (1). M o r e evidence emerged from comparison of pyrograms d e r i v e d from pure bacterial c e l l walls w i t h those d e r i v e d from w h o l e cells. 1

Current address: Emory University, Department of Chemistry, Atlanta, G A 30322.

0065-2393/83/0203-0705$06.00/0 © 1983 American Chemical Society

Craver; Polymer Characterization Advances in Chemistry; American Chemical Society: Washington, DC, 1983.


Tennessee Oranienberg

Typhimurium var. C o p e n h a g e n St. P a u l Choleraesuis var. K u n z e n d o r f Bareilly Montevideo Braenderup

Paratyphi A var. D u r a z z o Heidelberg Chester Derby Bredeney Paratyphi Β Java var. Odense

Species Name

6,7 6,7 m, t

Z29

y g, η ν s e, h

6,7 6,7 6,7

—

— e, m , z

1,5

1,7 1,2 (1, 2) 1>2 1,2 1,2 1,2 1,5

1, ν b b b

—

e, m , z —

1,2

Phase 2

e, h

i i e, h c

5, 12 4, 5, 12 4, 12, 27 4, 5, 12 4, 5, 12 4, 12

a r

Phase 1

Η Antigen (6)

1, 4, 5, 12 1, 4, 12 1, 4, 5, 12 6,7

4, 1, 1, 1, 1, 1,

2, 12 1, 4, 5, 12

Somatic (O) Antigen (6)

t

c,

Ci

c,

c

B B B

B B B B B B

A B

III III

III III III

XIV XIV XIV III

XIV XIV XIV XIV XIV XIV

XV XIV

Kauffmann (6) White Group Chemotype (7)

T a b l e I. Comparative Serology a n d C h e m i s t r y of Salmonella Species

Downloaded by UNIV OF TENNESSEE KNOXVILLE on November 14, 2016 | http://pubs.acs.org Publication Date: June 1, 1983 | doi: 10.1021/ba-1983-0203.ch040

6V2

2 2,4

2,4

2

2,4 2,4

2,4 2 2

2,4

a

Py/GC Characteristic Peaks

2

N > H O

n H

>

n

w

O r •
s, t

1, ν

d d (d) (d) d g, m

i d d

k

e, h d

r

k

1, w 1,6

1,5 1,6 1,7

1,6

1,5

1,5

6

1,5 1,5 1,2 1,5 1,5 z 1,2

4

2

3

D D D Ε E E E Ex G G G

D D D D D D D D D D

D

2

2

2

2

2

x

c c, c c c c c

XVI XVI XVI XIII XIII XIII XIII XIII VI VI VI

XVI XVI XVI XVI XVI XVI XVI XVI XVI XVI

III III XIV XIV XIV XIV XIV XVI


3 3 3

3 3 3 3 3 3 3 3 3 3

2 2 2 2 2 3


708

POLYMER CHARACTERIZATION

T h e pyrograms were very similar, but the former proved more d e f i n i tive i n their fingerprint characteristics (2). H o w e v e r , i n a study of Salmonella organisms an opposite c o n c l u s i o n was reached (3). T h e most important l i n e of evidence, of course, came directly from mass spectrometric (MS) identification of pyrolysis fragments. Simmonds (4) had d e d u c e d the probable macromolecular o r i g i n of fragments from the pyrolysis of two microorganisms (Table II). O t h e r studies (5, 6) have e n a b l e d investigators to catalog the i d e n t i f i e d frag ments from protein thermal decomposition and other sources of b i o logical macromolecules. There is considerable agreement on the i d e n tity of these macromolecule fragments. Before undertaking M S identification of pyrolysis fragments, certain aspects of the technique had to be evaluated. These i n c l u d e d variability i n c e l l cultures and gas chromatograph ( G C ) instrumental parameters. Experimental Cultured cells were washed three times in a small volume of distilled water. The heavy cell suspension was lyophilized and samples ranging from 40 to 120 μg were transferred to a tared quartz boat and weighed to the nearest microgram on an electrobalance. After completion of a pyrolysis run, the re sidual coke was weighed to determine the quantity of gaseous degradation products analyzed from each sample. Py/GC conditions have evolved over the years. A typical recent set of parameters is listed for a Chemical Data Systems Pyroprobe 100 Unit linked to a Varian 3700 G C . The G C was in turn coupled to a Varian M A T 112-S MS. The temperatures were as follows: pyrolyzer interface, 200 °C.; pyrolysis sample, 800 °C for 10.0 s. The temperature program began at 65 °C and was held for 4 min. It was then raised 6 °C/min to a final temperature of 165 °C., which was maintained isothermally. Recent work with a Carbowax 20M SCOT capillary column (43 m x 0.5 mm ID) had a resolution capability of 37,600 effective plates. Helium carrier gas velocity was measured at 38.5 cm/s. G C inlet and flame ionization detector (FID) temperatures were maintained at 250 °C. Electrometer amplifier was operated at a setting of 10~ amps full scale. The G C was connected to the MS by means of an open-split coupling. Interface and ion source temperatures were 250 °C. The electron beam was 80 eV and 1.5 mA. Ion accelerating voltage was 850 eV, resolution was 700, scan ning range was 1 s per mass decade. Spectra were acquired and stored by means of a Varian M A T S S 200 data system. In addition, exact mass mea surements of significant ions were obtained as well as chemical ionization spectra. Since undertaking these studies, we have examined several thousand samples of microbial and mammalian cells. From their pyrograms, we came to a number of general conclusions, some of which w i l l be discussed here. 12


40.

REINER AND MORAN


PylGC:

Biological Macromolecules

A Simple, Rapid

709

Technique

Samples are prepared by w a s h i n g and l y o p h i l i z i n g the cells. L y o p h i l i z e d cells store w e l l w i t h no discernable change w h e n e x a m i n e d over yearly intervals (7). O x b o r r o w c u l t u r e d cells on moistened filter paper are placed over the m e d i u m . Inoculating several different strains, e.g., Pseudomonas, on one paper membrane is feasible. T h i s method favors the i n c l u s i o n of metabolic products as w e l l as structural elements. C e l l s completely free of contaminating m e d i a were rem o v e d w i t h a spatula and aseptically d r i e d i n 7 m i n i n a stream of warm nitrogen (8,9). T h e technique must be rigorously standardized. P y / G C analysis time requires about 1 h to achieve a classification or identification. O r d i n a r i l y , m i n i m u m time from harvesting cells to e n d of analysis is about 2 h . B y traditional methodology, such identificat i o n w o u l d require several days. Pyrolysis Fragments Are

Immutable

T h e same structures were recognized i n the pyrogram of microorganisms e v e n w h e n operating parameters, c o l u m n , or different culture of the same strain of bacteria were used. T h e s e p h e n o m e n a have b e e n observed on numerous occasions. T h e avian and Battey strains of mycobacteria (now c o l l e c t i v e l y c a l l e d M. intracellare) gave characteristic triple e n d peaks that differed only i n amplitude. O v e r a p e r i o d of years (10—13), these characteristic e n d peaks were observed consistently and r e m a i n e d essentially unchanged. I n another case, the anaerobic organism causing b o t u l i s m can produce spores that can elaborate simple globular protein toxins. P y / G C e n a b l e d us to differentiate toxin types, and the toxic (proteolytic) strains proved to be different from nonproteolytic strains b y the increasing amplitude of three h i g h b o i l i n g compounds (a staircase effect) of the proteolytic strains. I n two laboratories, w i t h different instrumentation (capillary vs. packed column), and analyses performed 3 years apart, the staircase as a marker for proteolytic activity persisted (9, 14). Reproducibility

of PylGC

and PylGC IMS

Techniques

Pyrolysis techniques are so h i g h l y reproducible (15) that profiles can be superimposed to give essentially a single l i n e . I f operating conditions were closely controlled, relative standard deviations of retention times were usually i n the 0.2% range even after a 1-h recordi n g (14), and area measurements were i n the 8 - 2 0 % range. G o o d sample preparation, u n i f o r m , carefully controlled procedures, and



6

Ethanenitrile (9) A c r y l o n i t r i l e (9) Propanenitrile (10) Butanenitrile (12) 2-Methylpropanenitrile (13) M e t h y l b u t a n e n i t r i l e (15) M e t h y l p e n t a n e n i t r i l e (20) Benzonitrile (30) Phenylacetonitrile (39) T o l u n i t r i l e (40) P h e n o l (42) o-Cresol (41) p - C r e s o l (43) E t h y l p h e n o l (44) X y l e n o l s (45, 46) Indole (47) M e t h y l i n d o l e (48) Pyrrole (25)

Protein

c

c

c

c

Acrolein (l) Acetone ( l ) Butanone (4) Pentanone (8) Propanal ( l ) Methylpropanal (2) Methylbutanal (6) Furan (l) 2-Methylfuran (3) Dimethylfuran (7) F u r f u r a l (24) Methylfurfural (28) F u r f u r y l alcohol (33) Methylbutadiene

Carbohydrate

Table II. Assignment of Pyrolysis Fragments F

A c r y l o n i t r i l e (9) Ethanenitrile (9) Propanenitrile (10) Butanenitrile (12) P y r i d i n e (18) Methylpyridine

Nucleic acid c

c

Acrolein (l) Ethene ( l ) Propene ( l ) Butene ( l )

Lipid

c

c

3

1

4

Pyrrole (25) M e thy l p y rrole s (26, 27) Dimethylpyrroles (29, 31) C alkylpyrrole (34) C alkylpyrroles (36, 37)

Porphyrin

i in Both B. subtilis and M. luteus to Biological Classes'



c

c

c

c

c

(5) B e n z e n e (7)

c

a

A c e t a m i d e (35), p r o p i o n a m i d e (38), a n d a c e t o p h e n o n e (32) c o u l d not b e r e a d i l y a s s i g n e d to the classes l i s t e d a b o v e . * N u m b e r s i n p a r e n t h e s e s refer to p e a k n u m b e r o n c h r o m a t o g r a m (Ref. 4, p . 568). M a s s spectra w e r e r e c o r d e d e v e r y 2 s d u r i n g e l u t i o n o f the i n i t i a l c h r o m a t o g r a p h i c p e a k ( n u m b e r e d 1) w h i c h p e r m i t t e d i d e n t i f i c a t i o n o f i n d i v i d u a l c o m p o u n d s i n the m i x t u r e .

3

c

c

c

M e t h y l p y r r o l e s (26, 29) Methanethiol ( l ) Methane ( l ) Ethene ( l ) Propene ( l ) Butene ( l ) Methylpropene ( l ) Methylbutene ( l ) B e n z e n e (7) T o l u e n e (11) Styrene (21) E t h y l b e n z e n e (14) m-Xylene, p-xylene (15b) o - X y l e n e (17) P r o p y l b e n z e n e (16) C a l k y l b e n z e n e (19) P y r i d i n e (18) M e t h y l p y r i d i n e (22) D i n l e t h y l p y r i d i n e (23) E t h y l e n e oxide ( l )


712

P O L Y M E R

C H A R A C T E R I Z A T I O N

microprocessor instrumentation a l l contribute to this extraordinary reproducibility. These precise results have inevitably l e d several i n vestigators to consider a p p l y i n g computer methodology to the characterization of b i o l o g i c a l samples.


Computer Applications:

Cell Characterization

or

Identification

M e n g e r and coworkers (16) devised a simple program to differentiate pathogenic bacteria. T h e algorithm used was based solely on retention time and the area of a few key peaks of the profile. S i m i l a r attempts have b e e n undertaken by others (17—21). A scheme for automatic identification or classification of cells from their recorded mass pyrograms has b e e n conceived (22). B r i e f l y , the scheme calls for establishing a reference pattern for each c e l l type. U n k n o w n samples w o u l d be compared systematically to appropriate sets of mass pyrograms. F i n a l l y , statistical techniques w o u l d be i n v o k e d to arrive at a diagnosis. Schafer also describes a t i m e - w a r p i n g function w h i c h permits alignment of mass pyrograms recorded under different operating conditions (22). Microbial

Studies

Heretofore, c e l l characterization has b e e n accomplished by s i m ple visual comparison of test and reference pyrograms. It is perhaps fortuitous that simple diagnostic information can be obtained from pyrolysis of an extremely complex matrix. I n contrast to nonpyrolytic chromatographic studies recorded i n the literature, a l l of our investigations except those u t i l i z i n g primary mammalian cells have b e e n completed w i t h c o d e d samples. O n e notable study i n v o l v e d double b l i n d samples of h u m a n tuberculosis and other mycobacterial cells. T h i s was the extent of the information prov i d e d to the analysts, w h o were g i v e n no clues w i t h regard to w h i c h of the 50 samples were of the same or different strain, h o w they were grown, how inactivated, etc. T h e cells were subjected to P y / G C on two different instrumental systems and each sample was analyzed at least twice. E x c e p t for some uniform variation i n retention t i m e , the two sets of profiles were remarkably similar. B y laborious v i s u a l comparisons of over 200 pyrograms, we were able to select certain key peaks i n the profiles that we considered to be significant. Pyrograms were sorted into five groups of 10 samples each. T h e criteria for differentiation, the key peaks, p r o v e d to be a simple process once a pattern was recognized. F o r example, l y o p h i l i z e d cells of Mycobacterium tuberculosis always y i e l d e d a pyrogram h a v i n g a short peak flanked b y two peaks of greater amplitude i n a specific retention time b a n d . T h e relationship always h e l d . W h e n the decoding took place, it was learned that a l l 48 cultures



40.

REINER

A N D M O R A N

Biological

Macromolecules

713

had b e e n classified correctly—or i d e n t i f i e d i f reference profiles had b e e n available. (We had i n d i c a t e d to the bacteriologist that two of the cultures had g i v e n bizarre profiles; on examination, it was l e a r n e d that these two came from tubes contaminated by a mold.) T h e work on mycobacterial identification has great practical significance. F o r those suspected of harboring the tuberculosis b a c i l l u s , a diagnosis can take from 3 to 6 weeks or more. (The organism is slow growing.) I n only 2 weeks' subculture, P y / G C enabled the analyst to make a definitive diagnosis. Moreover, essentially the same patterns were p r o d u c e d w h e n c u l t u r e d i n three different m e d i a . T h i s p h e n o m e n o n appears to be u n u s u a l because most workers report that each m e d i u m gives it o w n pyrogram pattern. T h e mycobacteria, w h i c h i n c l u d e the leprosy b a c i l l u s , are u n i q u e i n their c e l l w a l l structure, some 4 0 % or more b e i n g of complex l i p i d . Some of these, the m y c o l i c acids, have chains or rings n u m b e r i n g 88 carbon atoms. Structural studies are still incomplete but excellent P y / G C / M S investigations on m y c o l i c acids have b e e n carried out (23). Another study of pathogenic bacteria p r o v e d to be interesting. T h i s study i n v o l v e d the enteric organisms, Salmonellae, w h i c h c o m m o n l y cause food poisoning. Fifty-four of the most c o m m o n l y e n countered (coded) strains were subjected to P y / G C . T h e y were correctly sorted and classified. Results are summarized i n T a b l e I (24). I n most studies, P y / G C differences are attributed to changes i n relative peak height. I n this study, however, some of the strains were characterized by the presence or absence of peaks. O f special interest was the close correlation b e t w e e n antigenic structures and u n i q u e pyrolysis peaks. T h e pyrolysis peaks also showed a close relationship w i t h the so-called chemotypes. T h e chemotypes are unusual dideoxy hexoses such as paratose, abequose, and tyvelose sugars that are found i n their c e l l walls (25). T h e s e sugars are considered to be prominent markers. T h e pyrolysis technique, w h e n a p p l i e d to b i o l o g i c a l macromolecules, can b r i n g out small yet significant marker peaks. I n a different context, the neurotransmitter, acetylcholine, has b e e n isolated from neural tissue (26). D i p i c o l i n i c a c i d has served as a marker for the bursting forth or release of bacterial spores (8). I n a most i n teresting study d e a l i n g w i t h pyrolysis of enzymes, four major peaks were tentatively i d e n t i f i e d as compounds d e r i v e d from tyrosine and tryptophan (27). Mammalian

Cells

Several investigations were undertaken on both c u l t u r e d and primary cells. P y / G C profiles were recorded for mouse a n d rabbit k i d n e y and red b l o o d cells, a n d for normal and l e u k e m i c w h i t e b l o o d



714


cells. I n addition, carcinogenic experiments were performed on C h i n e s e and Syrian hamster cells (28)? R e l i a b l e fingerprinting, i.e., differentiation, was a c h i e v e d i n those studies and lends some encouragement to the proposition that c h e m i c a l differences b e t w e e n normal and cancer cells do exist. D u r i n g this p e r i o d , another l i n e of investigation was f o l l o w e d . W e r e c e i v e d a batch of c u l t u r e d cells d e r i v e d from h u m a n patients afflicted w i t h various i n h e r i t e d b i o c h e m i c a l disorders. These cells (fibroblasts) were examined by P y / G C and their profiles showed distinct differences, although the G C columns used at that time were quite inefficient by present day standards (29). W e reported on a study of c u l t u r e d fibroblasts from patients w i t h the i n h e r i t e d disease, cystic fibrosis ( C F ) (30). C F is an autosomal genetic disease w h i c h afflicts one out of every 2000 l i v e births among Caucasians. T h e cause of the disease is u n k n o w n . I f hétérozygotes (gene carriers, but the i n d i v i d u a l s themselves are not afflicted) c o u l d be designated, such designation w o u l d constitute a major advance i n diagnosis. T h e pyrograms d i d i n d e e d show slight but repeatable differences b e t w e e n a heterozygous father and his two C F sons. T h e pyrograms were compared to that of a normal female subject. A l l of the C F c u l tured cells appeared to have the same profile characteristics. Results on a l i m i t e d n u m b e r of samples (nine) were encouraging, and future work i n this area is contemplated u t i l i z i n g the c o m b i n e d P y / G C / M S system. T h e first report on P y / G C / M S of normal h u m a n tissue cells appeared i n 1979 (22). I n this work, frozen, unfixed samples of l i v e r , brain, spleen, and k i d n e y taken from recent accident victims were converted into analytical samples. T h e conversion consisted m e r e l y of g r i n d i n g small portions of tissue i n d i s t i l l e d water and l y o p h i l i z i n g the resultant suspension. Results of this investigation brought some interesting facts to light. T h e four tissues c o u l d be clearly differentiated and, quite unexpectedly, the same tissue obtained from different i n d i v i d u a l s gave very similar profiles. M o l e c u l a r pyrolysis products, 44 i n number, were i d e n t i f i e d by M S . N o t surprisingly, many of the i d e n t i f i e d fragments have b e e n found i n microorganisms and e v e n i n geological samples. Single and m u l t i p l e i o n mass chromatographic techniques were also used to exploit the characteristic differences b e t w e e n tissue specimens. C o n c e i v a b l y , i n the future, baseline information such as that recorded i n this study c o u l d be used to discriminate normal and pathological cells. The caption of Figure 3, Reference 28, should read " C h . Hamster—Normal Tumor Cell Line." 2



40.

REINER AND MORAN

Biological

• I

20

715

Macromolecules

I • • •

•

I . . . .

40 T i m e (Minutes)

ι . . . .

I ..

1 60

. ι ι ι I

Figure 1. Py/GC chromât ο grams of capsular typable and nontypahle strains of K. pneumoniae. Key: A, C2; B, C6; and C., CI. Key to peak identification: 1, acetonitrile; 2, toluene; 3, ethylhenzene; 4, pyridine; 5, pyrrole; 6, furfuryl alcohol; 7, phenol; and 8, cresol. Epidemiological Studies P y / G C / M S can be used i n a practical sense for solving epidemiolog ical problems (31). A n u m b e r of samples of Klebsiella pneumoniae isolated from hospital-acquired infections p r o v e d to be untypeable b y the usual serological methods. H o w e v e r , nine c o d e d duplicate strains of capsular nontypeable K. pneumoniae were analyzed a n d correctly matched by P y / G C / M S . Pyrograms of three of the cultures are shown i n F i g u r e 1.


716


T h e large peak that appears i n F i g u r e I C at a retention time of 12 m i n , and precedes the prominent pyrrole peak (4), has b e e n i d e n t i f i e d as acetic acid. T h i s latter peak serves as an important marker for dif ferentiation of several Klebsiella strains. Assignment of the eight compounds to their probable macromolecular origin can be made b y referring to T a b l e I.


Conclusions Pyrolysis techniques have great potential as a means of o b t a i n i n g important information on b i o l o g i c a l macromolecules. T h i s potential extends to both pure research and practical, nonresearch applications. I n the future it is conceivable that pyrolysis techniques c o u l d form a n e w system of taxonomy—a chemotaxonomy. S u c h a scheme c o u l d be a valuable adjunct to classical methods of classifying flora, fauna, a n d geological samples. Acknowledgments T h e authors w o u l d l i k e to thank E d g a r R i b i of the N I H R o c k y M o u n t a i n Laboratory w h o k i n d l y s u p p l i e d the pure c e l l w a l l samples for this study. Literature Cited 1. Salton, M. R. J. In "The Bacterial Cell Wall"; Elsevier: Amsterdam, 1964; pp. 133-85. 2. Reiner, E., unpublished data. 3. Emswiler, B. S.; Kotula, A. W. Appl. Environ. Microbiol. 1978, 35, 97. 4. Simmonds, P. G. Appl. Microbiol. 1970, 20, 567. 5. Merritt, C., Jr.; Robertson, D. H. J. Gas Chromatogr. 1967, 5, 96. 6. Stack, M. V. J. Gas Chromatogr. 1967, 5, 22. 7. Reiner, E . ; Ewing, W. H. Nature 1968, 217, 194. 8. Oxborrow, G. S.; Fields, N. D.; Puleo, J. R. In "Analytical Pyrolysis"; Elsevier: Amsterdam, 1977; p. 69. 9. Reiner, E. In "Analytical Pyrolysis"; Elsevier: Amsterdam, 1977; p. 49. 10. Reiner, E. Nature 1965, 206, 1272. 11. Reiner, E. J. Gas Chromatogr. 1967, 5, 65. 12. Reiner, E.; Kubica, G. P. Am. Rev. Respir. Dis. 1969, 99, 42. 13. Reiner, E.; Beam, R. E.; Kubica, G. P. Am. Rev. Respir. Dis. 1969, 99, 750. 14. Reiner, E.; Bayer, F. L. J. Chromatogr. Sci. 1976, 16, 623. 15. Irwin, W. J.; Slack, J. A. The Analyst 1978, 103, 673. 16. Menger, F. M.; Epstein, G. Α.; Goldberg, D. Α.; Reiner, E . Anal. Chem. 1972, 44, 423. 17. Carmichael, J. W.; Sekhon, A. S.; Sigler, L. Can. J. Microbiol. 1973, 19, 403. 18. Macfie, H. J. H.; Gutteridge, C. S.; Norris, J. R. J. Gen. Microbiol. 1978, 104, 67. 19. Eshuis, W.; Kistemaker, P. C.; Meuzelaar, H. L. C. In "Analytical Pyro lysis"; Elsevier: Amsterdam, 1977; p. 151. 20. Schulten, H. R.; Beckey, H. D.; Meuzelaar, H. L. C.; Boerboom, A. J. H. Anal. Chem. 1973, 45, 191. 21. Kistemaker, P. G.; Meuzelaar, H. L. C.; Posthumus, M. In "New Ap


40.

22. 23. 24. 25. 26.


27. 28. 29. 30. 31.

REINER AND MORAN

Biological Macromolecules

717

proaches to the Identification of Microorganisms"; Heden, C.; Illeni, T., Eds.; Wiley: New York, 1975; p. 179. Reiner, E . ; Abbey, L . E . ; Moran, T. F.; Papamichalis, P.; Schafer, R. W. Biomed. Mass Spectrom. 1979, 6, 491. Etemadi, A. H.; Miquel, A . - M . ; Lederer; Barber, M . Bull. Soc. Chim. Fr. 1964, 3274. Reiner, E . ; Hicks, J. J . ; Ball, M . M.; Martin, W. J . Anal. Chem. 1972, 44, 1058. Luderitz, O.; Staub, A. M.; Westphal, O. Bacteriol. Rev. 1966, 30, 192. Schmidt, D. E . ; Szilagyi, P. I. Α.; Alkon, D. L . ; Green, P. Science 1969, 165, 1370. Danielson, N . D.; Glsjch, J. L . ; Rogers, L . B. J. Chromatogr. Sci. 1978, 16, 455. Reiner, E.; Hicks, J. J. Chromatographia 1972, 5, 525. Reiner, E . ; Hicks, J. J. Chromatοgraphia 1972, 5, 529. Reiner, E . J.; Moran, T. F. J. Chromatogr. (Biomed. Appl.) 1980, 221, 371. Abbey, L . E . ; Highsmith, A. K.; Moran, T. F.; Reiner, E . J. J. Clin. Mi crobiol. 1981, 13, 313.

RECEIVED for review October 14, 1981. ACCEPTED March 2, 1982.


Mass Spectrometry of Biological

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