Gas-liquid chromatography-mass spectrometry of carbon-13 enriched amino acids as trimethylsilyl derivatives

251

BIOCHIMICAET BIOPHYSICAACTA BBA 26318

G A S - L I Q U I D CHROMATOGRAPHY-MASS S P E C T R O M E T R Y OF CARBON-I3 E N R I C H E D AMINO ACIDS AS T R I M E T H Y L S I L Y L D E R I V A T I V E S W. J. A. VANDENHEUVEL AND JACK S. COHEN* Merck Sharp and Dohme Research Laboratories, Rahway, N. J. (U.S.A.)

(Received October 23rd, 1969)

SUMMARY Combined gas-liquid chromatography-mass spectrometry of 13C enriched amino acids as their trimethylsilyl derivatives is reported. The 13C content of the amino acids and various fragments are calculated from relative peak heights in the mass spectra. Comparison of the values obtained indicates a 13C biosynthetic isotope effect. Characteristic relative intensities for certain fragment ions permit structural assignments. No evidence for gas-liquid chromatographic fractionation of the normal and corresponding 13C enriched amino acids was observed.

INTRODUCTION We wish to report on the gas-liquid chromatography-mass spectrometry of 13C enriched amino acids. These were isolated from algae grown on 1~C enriched (15%) CO~ following hydrolysis and preparative column chromatography 1. 13C magnetic resonance data for the amino acids are presented elsewhere *. Although amino acids themselves are not amenable to gas-liquid chromatographic analysis, satisfactory volatility is found for the n-butyl-N-trifluoroacetyl estersS, 4 and the O,N-ditrimethylsilyl derivativesS-L the latter of which were used in the present work. Initial stages of our investigation involved the use of gas-liquid chromatography to provide information concerning the composition of the fractions obtained by ionexchange chromatography. Satisfactory separation of pairs of amino acids not readily differentiated b y thin-layer chromatography (for example, serine and threonine, and leucine and isoleucine) was obtained, as expected 5-7. The combined gas-liquid chromatography-mass spectrometry technique4, 8-1. is a very efficient means for the identification of sub-milligram quantities of compounds in mixtures, as well as allowing estimation of isotopic content. Further, the presence of isotopic labels can facilitate the assignment of structures to fragment ions formed during low resolution mass spectrometry. We have employed the combined technique * Present adress : Division of Computer Research and Technology, National Institutes of Health, Bethesda, Md., U.S.A. Biochim. Biophys. Acta, 2o8 (197o) 251-259

252

W . J . A . VANDENHEUVEL, J . S . COHEN

to investigate the mass spectrometric behavior of the trimethylsilyl derivatives* of the 1~C enriched amino acids. M A T E R I A L S AND METHODS

Combined gas-liquid chromatography-mass spectrometry was effected with the LKB Model 9ooo instrument. Two spiral glass columns were employed, 9 It (2% SE-3o ) or 6 ft (2% OV-IT)×3.5 mm internal diameter (3o ml/min). A variety of column temperatures were employed (7o-I6O °) because of the wide range of volatilities of the amino acid derivatives. Mass spectra were obtained using 7 ° eV ionizing potential; 27 °o ion source temperature; 5o #A filament current; 3.5 kV accelerating potential. Several scans (4-8 sec) were taken during or near the period of maximum gas-liquid chromatographic peak height; no serious mass spectrometric "bias ''l~ due to concentration changes was observed. Trimethylsilyl derivatives of the amino acids were prepared following the method of STALLINGet al. s. RESULTS AND DISCUSSION

The mass spectrum of the trimethylsilyl derivative of glycine obtained using gas-liquid chromatography-mass spectrometry is presented in Fig. I. No molecular ion (M)** is observed at m/e 219, but a fragment ion corresponding to loss of a methyl group (m/e 204) is observed, not unusual for trimethylsilyl derivatives n-13. The signal at isotope peak m/e 205 ( S + I ) represents the loss of a methyl group from the glycine

,ooj 90

102

80

70

5c X5

~

~: 4c

204

/76

l0 1

o--~ 20

~L .,I.,[ 50

I

75

h

i

4. I I00

~ 125

150

175

200

225

m/e

Fig. I. M a s s s p e c t r u m of t h e d i - t r i m e t h y l s i l y l d e r i v a t i v e of g l y c i n e .

* A f t e r t h e p r e p a r a t i o n of t h i s m a n u s c r i p t BAKER et ctl. 13 r e p o r t e d o n t h e m a s s s p e c t r o m e t r y of s e v e r a l a n f i n o a c i d s a n d di- a n d t r i - p e p t i d e s as t h e i r t r i m e t h y l s i l y l d e r i v a t i v e s . ** T h e i o n f r o m a m o l e c u l e h a v i n g t h e s a m e m a s s as t h e m o l e c u l a r w e i g h t is r e f e r r e d t o as t h e " m o l e c u l a r i o n " , or " M " , r a t h e r t h a n " p a r e n t p e a k " , or ,,p,,14. B i o c h i m . B i o p h y s . Mcta, 2o8 (197 o) 2 5 1 - 2 5 9

253

ANALYSIS OF 13C ENRICHED AMINO ACIDS

di-trimethylsilyl molecules of I m/e unit higher mass*. This peak arises from the presence of molecules containing one atom of naturally occurring laC, *H, 29Si, 15N or 170. The mass spectrum of the di-trimethylsilyl derivative of ~3C enriched glycine is presented in Fig. 2. These two compounds differ only in their ~"C content, and hence the observable differences in their peak intensities are a direct reflection of the extent of incorporation of ~3Cinto the amino acid. The mass spectra of the di-trimethylsilyl derivatives of phenylalanine and the 13C enriched phenylalanine are shown in Figs 3 IOC

9C

102

8C

z

5(

X5

~204 176

147

3C

r

2c

i,L, .IL [ 20

50

I h

,.

1

75

1

125

150

175

200

225

m/e

Fig. 2. Mass spectrum of the di-trimethylsilyl derivative of 1~C enriched glycine. 73

tO0

/92 70

~ ~° ~ 5o ,~

266 xlO

~'40

30

294

2C

'l'

20

l

50

J, i

l

,L

i

75

~

I00

•

. . l

125

l

I

[, ,

IB ~

150

l

I

, ~75

I

r

I

!

'~

200

l

2251 I

;

250

I

l 275

I '3 0I 0 l

m/e

Fig. 3. Mass spectrum of the di-trimethylsilyl derivative of phenylalanine. * W e assume that contributions of M - - 1 4 (CH2) to the i n t e n s i t y of m/e 205 are negligible;such a f r a g m e n t a t i o n has not been reported after e x t e n s i v e mass spectrometric experience with trimethylsilyl derivatives of m a n y compounds.

Biochim. Biophys. Acta, 208 (197 o) 251-259

254

w.j.A.

VANDENHEUVEL, J. s. COHEN

7,5

I00

7O

60 _z w

~_50 4C

XlO 266~,

J

.I.

, d i

5O

75

L.

~

I00

'

' l 125

J l

L

,I . . 150

1,1' .

. . 175

--

200

i '

' 225

. . . . . . ~50 I

\

i 1~7~

'i r

300

role

Fig. 4. Mass s p e c t r u m of the di-trimethylsityl derivative of z3C enriched phenylalanine.

and 4, respectively. The molecular ion (role 309) is again absent, but, as with glycine, a (M--15) fragment is seen. The relative intensity of the (S + I) peak in general increases with increase in the overall 13C percentage, i.e. the probability of a fragment containing I lsc atom is related to its ( S + I ) peak intensity. Let AI1 represent the difference between the intensities of the ( S + I ) peaks of the ~sC enriched and the normal amino acids, with the intensity of the S peak, Is, set equal to IOO in each case. Then, clearly, the ratio of the difference in intensities of the (S + i) to the S peak, i.e. AI~/IOO, will give a measure of the overall lsC content. However, for the same overall 13C percentage the probability of any molecule containing I 13C atom will increase with the number of carbon atoms in the molecule. This fact is reflected in the greater relative intensity of the ( S + I ) peak for the (M--I5) fragment of phenylalanine compared to that of glycine (Figs. 4 and 2, respectively) is. I t can be shown that IOO ~'C02 C --

-

-

(I)

where, r = AI1/IOO, m is the total number of amino acid carbon atoms in the molecule or fragment,C0 is the natural abundance of 12C(98.9), and c is the 13C content expressed as excess atom %. The percent natural abundance of 13C (I.I) must be added to this to obtain the total percentage, since it has been subtracted when correcting for the normal amino acid, whose ( S + I ) peak also contains contributions from ~Si, etc. For example, consider the (M--I5) fragment of glycine, with I s set equal to IOO in both cases: normal glycine, I(s+ 1) = 18; 1~C enriched glycine, Iis+l) = 49, All = 3I, r = o.31 ; therefore, c = 13.4 and total percent 1~C = 14. 5. Table I presents the total 13C content for fragment ions of each of the enriched amino acids. In the two cases of methionine and aspartic acid both M and (M--I5) fragments were observed whereas for the other amino acids (except lysine (TMSi)3, which shows no (M-I5)) only the latter is noted and the 13C percentage obtained for Biochim. Biophys. Acta, 208 (197 o) 251-259

ANALYSIS OF laC ENRICHED AMINO ACIDS

255

TABLE I 13C CONTENT OF THE AMINO ACIDS

Amino acid

Fragment

role

Number of amino acid C atoms

18C ( % )

Gly

M--I 5 M--43 M--II 7

204 176 lO2

2 i I

14.2 i5. 3 14.6

Ala

M--II 7

116

2

14.6

Set

M--I 5 M--43 M--II 7 M--R

3 °6 278 204 218

3 2 2 2

13. I 13.1 13.1 13. 9

Thr

M--I5

320

4

13.7

Asp

M M--I5 M--43 M--II7 M--R

349 334 3°6 232 218

4 4 3 3 2

14.2 14.2 14.4 14.1 13. 9

Pro

M--I 5 M--43 M--II7

244 216 142

5 4 4

13. I 13.7 13.5

Val

M--I5 M--II7

246 144

5 4

13.6 13.2

Met

M M--I5 M--43 M--II7

293 278 250 176

5 5 4 4

14.o 14.o 13.5 13.7

Leu

M--I 5 M--43 M--II7 M--R

26o 232 158 218

6 5 5 2

13.3 13.o 13.6 14.2

Ile

M--I5 M--43 M--II7

260 232 158 218

6 5 5 2

13. 3 13. I 13.3 13. 9

His

M--IS M--II7 M--R

356 254 218

6 5 2

12.9 12.8 13. 9

Lys (TMSi)3

M

362

6

12. 9

Lys (TMSi),

M--II 7

317

5

13.7

Phe

M--IS M--43 M--II7 M--R

294 266 192 218

9 8 8 2

12.7 12.8 12. 4 14.2

Tyr

M--I5 M--43 M--II 7 M--R

382 354 280 218

9 8 8 2

12.4 12. 7 12. 5 14.2

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each are consistent. Since with methionine the same value is observed for both the M and (M--I5) fragments. Another frequently observed fragment is (M--43), which appears to be (M-- (15 +28)), loss of methyl and CO with retention of the silyloxy group. A third common pathway is M - - I I 7, loss of carbotrimethylsilyloxy; a similar fragmentation has been observed b y HORNING et al. 16 with trimethylsilyl derivatives of ~-keto acids. This is analogous to the loss of 45 (COOH) from amino acids and loss of 73 (COOC~H~) and IOI (COOC,Hg) from amino acid esters4,17,18. Many of the amino acids yield a fragment at role 218, which results from loss of the amino acid side chain 7; an analogous fragmentation is observed with free amino acids~4O8. Other commonly observed fragment ions are role 73 and 147, [Si(CHa)31+ and [(CH3)a-SiO--Si(CH3)2~ +, respectively 13. Most of these fragmentations can be seen in the mass spectra of trimethylsilyl glycine and phenylalanine. The m/e 218 fragment is useful in that it enables a value for the laC content to be calculated which is independent of the nature of the side-chain, and as seen from Table I, values for these fragments are consistent. (Calculation of the 1~C content from this fragment is difficult for several amino acids (e.g. methionine and threonine) which produce interfering fragments of m/e 219). Using Eqn. I to calculate the 13C content for nearly all the amino acid fragments, the values obtained (Table I) show a spread which depends on the size of the fragment. This is illustrated b y calculation of standard deviations for each group of fragments, Number of amino acid C atoms

Total 13C ( % )

Number of values used

1,2 3,4 5,6 8,9

14.2 13. 7 13.3 12.6

13 12 15 6

± ± ± 4-

0.4 0. 4 0.4 0.2

While these differences are small, and there is in fact considerable overlap, the trend is nonetheless real. I t cannot simply be explained in terms of the larger molecules having less overall 13C content, since the role 218 fragments derived from them (e.g. phenylalanine, tyrosine) give the higher values. Thus, the differences would appear to be in the side-chain group, and can only be explained in terms of a small isotopic biosynthetic preference for 12C for these residues. No explanation involving the growth or chemical treatment of the algae 1, nor of the nature of the gas-liquid chromatography mass spectrometry method (see below) or calculation would seem to be forthcoming. A number of 14C and 13C isotope effects in biological systems have previously been described (for a tabulation see ref. I9), although in these cases determinations of isotopic content were made (using radioactivity for 14C and mass spectrometry for 13C) on bulk CO2. An isotope effect has also been reported for a number of separated ~*C labeled amino acids derived from algae, b y measurement of their specific activities 2°. The effect was also greatest in this case for phenylalanine and tyrosine, as might be expected from considerations of the relative number of enzymatic steps at which potential isotope effects can occur in the biosynthesis of the amino acids 21. Also, the ratio of the 14C/12C effect reported (330/0)20 and the m a x i m u m 1~C/1~C effect (about 19%) presented here is about 2, as expected for comparison of the same overall Biochim. Biophvs. Acta, 208 (197 o) 251-259

257

ANALYSIS OF laC ENRICHED AMINO ACIDS

process19, 2z. In another study the carboxyl groups of the separated amino acids (as 14CO~) were found to be uniformly labeled 23, which is consistent with our finding for the m/e 218 fragments. The observation of differential isotope effects in separated components of biological systems, by the use of stable isotopes and mass spectrometry, could have considerable value for detailed studies of metabolic pathways. This may be illustrated by the recent reports of BOSE et al.2~,25 on the biosynthesis of gliotoxin in which incorporation from a series of labeled precursors was determined. Fragment ions, with the same number of amino acid carbon atoms would be expected to exhibit similar and characteristic values for AI1 (expressed as RIV, see Table II, and it can be perceived that the (M--I 5 fragmentation involves the TMSi group and not an acid methyl group. It is only necessary to compare the relative intensity value for the (M--I5) fragment of alanine (role 218) to any other role 218 value (Table II) to discern that the methyl group eliminated does not come from alanine itself. Also, the relative intensity value for a (M--II7) fragment is reduced from that of the corresponding M or (M--I5) fragment by a quantity TABLE II CHARACTERISTIC RELATIVE INTENSITY"VALUES FOR THE ( S - - I ) PEAKS OF 13C ENRICHED AMINO ACID IONS Relative i n t e n s i t y value (RIV) = IooAIl/(Ioo+AI1) ; see text. M indicated b y +.

Number of C atoms from amino acid moiety

M or ( M - - r S ) RIV AA role

(M--43) RIV AA

role

(M--zr7) RIV AA

m/e

15

Gly

2

24

Gly

2o 4

23 23

Ser Ala

176 278 19o

14 23 24

Gly Ser Ala

lO2 2o 4 116

3

3° 3° 38 38 37 43 43 41 42 46 46 45 45

Set Ala Asp Asp Thr Met Met Pro Val Leu Ile His Lys

3o6 218"* 349 + 334 320 293 + 278 244 246 26o 260 356 362+,§

32

Asp

3o6

31

Asp

232

37 34

Met Pro

25o 216

41 41

Leu Ile

232 232

37 37 36 42 42 41 42

Met Pro Val Leu Ile His Lys

176 142 144 158 158 254 317 ***

52 52

Phe Tyr

266 354

51 51

Phe Tyr

192 28o

I

4

5

6

8 9

55 54

Phe Tyr

218 -RIV

AA

23 23 24 23 23 24 24 25

Ser Asp Leu Ile His Phe Tyr Val*

294 382

* Loss of R group isopropyl, r a t h e r t h a n M - - ( I 5 + 2 8 ) , is p r o b a b l y p r e d o m i n a n t p a t h w a y leading to formationL see text. * * Appears to result from loss of CH 3 from trimethylsilyl group r a t h e r t h a n R group CH3; see text. *** F r o m Lys(TMSi)4. § Lys (TMSi)v

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just sufficient to indicate that the (M--II7) fragment contains one less amino acid carbon atom than the molecular ion. The same holds for the (M--(I5+28)) fragments. Good correlation of relative intensity value with number of amino acid carbon atoms is indicated in Table II. This allows tentative assignment of structure in those instances in which fragment ions could arise via several of the different proposed pathways. For example, the role 218 fragment of valine could arise from either (M--(I5+28)) to produce a species containing four carbon atoms originating from the amino acid itself, or from loss of the isopropyl group to yield the commonly observed role 218 containing two amino acid carbon atoms. The main pathway appears to be the latter, for the value of 25 in Table II is much closer to that expected for a two carbon rather than a four carbon amino acid fragment. A recent study employing multi-scan gas-liquid chromatography-mass spectrometry demonstrated conclusively that partial isotopic fractionation does occur for the trimethylsilyl derivatives of/3-D-glucose and/5-D-[2H7~glucose with a 2-m column, the mass spectra changing with time across the gas-liquid chromatographic peak ~8. Isotope fractionation during gas-liquid chromatography would complicate our estimation of the 13C content of the amino acids, since the calculation depends upon the intensity of the m/e signals at S and (S+I). We assume that the mass spectral isotope cluster intensity values are constant with time, i.e. that the gas-liquid chromatographic peak is homogeneous with respect to isotope composition. No isotope fractionation was indicated in this case from a comparison of retention times, peak shapes and theoretical plate values. Lack of fraetionation was confirmed by multiple mass spectral scans in the region of M and (M--I5) across the gas-liquid chromatographic peaks of a number of the I3C enriched amino acids; no significant variation in the relative intensities of isotope peaks for these fragments was found with respect to time of scan. This lack of isotope fractionation for the I3C containing amino acids is perhaps not surprising, since the conditions (I5-m column) BENTLEY et al. ~7 found effective in resolving the trimethylsilyl derivatives of glucose and E~H7]glucose failed to separate a mixture of the trimethylsilyl derivatives of glucose and uniformly labeled I13C]glucose. ACKNOWLEDGMENTS

We would like to thank W. Horsley and H. Sternlicht for supplying the 13C enriched algae, R. P. Buhs and O. C. Speth for technical suggestions, and J. L. Beck, E. Gilbert, J. L. Smith and H. M. Fales for helpful discussions. REFERENCES i 2 3 4 5 6 7 8 9 io ii

J. s. COHEN, W. HORSLEY AND H. STERNLICHT, in preparation. W. HORSLEY, n . STERNLICHT AND J. s. COHEN, Biochem. Biophys. Res. Commun., 37 (1969) 47. D. ROACH AND C. W. GEHRKE, J. Chromatog., 43 (1969) 303 • E. GELPI, W. A. KOENIG, J. GILBERT AND J. ORO, J. Chromatog. Sci., 7 (1969) 6°4. D. L. STALLING, C. W. GEHRKE AND R. W. ZUMWALT, Biochem. Biophys. Res. Commun., 3I (1968) 616. F. SHAKROKHI ANn G. W. GEHRKE, J. Chromatog., 36 (1968) 31. A. E. PIERCE, Silylation of Organic Compounds, Pierce Chemical Co., Rockford, II1., 1968, p. 38. R. RYHAGE, Anal. Chem., 36 (1964) 759. J- T. WATSON AND K. BIEMANN, Anal. Chem., 36 (1964) 1135. F. A. J. M. LEEMANS AND J. A. McCLOSKEY, J. Am. Oil Chemists' Soe., 44 (1967) i i . C. J. W. BROOKS, E. C. HORNING AND J. S. YOUNG, Lipids, 3 (1968) 391.

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12 E. C. HORNING, C. J. W. BROOKS AND W. J. A. VANDENHEUVEL, in 1R. PAOLETTI AND D. KRITCHEVSKY, Advances in Lipid Research, Vol. 6, Academic Press, New York, 1968. 13 K. M. BAKER, M. A. SHAW AND D. H. WILLIAMS, Chem. Commun., (1969) 11o8. 14 K. BIEMANN, Mass Spectrometry, Organic Chemical Applications, McGraw-Hill, New York, 1962, p. 44. 15 J. H. BEYNON AND A. E. WILLIAMS, Mass andAbundance Tables for Use in Mass Spectrometry, Elsevier, A m s t e r d a m , 1963, p. v i i ; w . J. VANDENHEUVEL, J. L. SMITH AND J. S. COHEN, in Proe. 6th Intern. Symposium on Advances in Chromatography, M i a m i Beach, Florida, June

z97o. 16 M. G. HORNING, E. A. BOUCHER, A. M. MOSS AND E. C. HORNING, Anal. Letters, I (1968)713 . 17 K. BIEMANN, Mass Spectrometry, Organic Chemical Applications, McGraw-Hill, New York, 1962, C h a p t e r 5. 18 I~. BIEMANN AND J. A. McCLOSKE¥, J. Am. Chem. Soc., 84 (1962) 3192. 19 V. F. RAAEN, G. A. ROPP AND H. A. ]~AAEN, Carbon-r 4, McGraw-Hill, New York, 1968, p. 83. 20 J. R. CATCH, Proe. Radioisotope Conf., 2nd, Oxford, 5954, Vol. I, B u t t e r w o r t h , London, 1954, p. 258. 21 J. R. MATTOON, in P. BERNFELD, The Biogenesis of Natural Compounds, Pergamon, New York, 2nd ed., 1967. 22 D. L. BUCHANAN,A. NAKAO AND G. EDWARDS, Science, 117 (1953) 541. 23 !K. H. HALLOWES, F. P. W. WINTERINGHAM AND g . J. LEQUESNE, Nature, 181 (1958) 336. 24 A. K. BosE, K. G. DAS, P. T. FUNKE, I. KUGAJEVSKY,P. O. SHUKLA, K. S. KHANCHANDANI AND R. J. SUHADOLNIK,J. Am. Chem. Soc., 90 (1968) lO38. 25 A. K. BOSE, I~. S. I~HANCHANDANI,R. TAVARES AND P. T. FUNKE, J. Am. Chem. Soc., 90 (1968) 3593. 26 C. C. SWEELEY, W. H. ELLIOTT, I. FRIES AND R. RYHAGE, Anal. Chem., 38 (1966) 1549. 27 R. BENTLEY, N. C. SAHA AND C. C. SWEELEY, Anal. Chem., 37 (1965) 1118.

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