842 NUCLEIC ACID CONSTITUENTS [46]

Acknowledgments

The author is indebted to many colleagues who contributed to this work over a period of time, including K. J. Lyman, B. Basile, H. Pang, K. H. Schram, and S. C. Pomerantz. The methods described were developed and supported by NIH grants GM 21584 and CA 18024.

[46] Gas C h r o m a t o g r a p h y - M a s s S p e c t r o m e t r y of F r e e

R a d i c a l - I n d u c e d P r o d u c t s o f P y r i m i d i n e s a n d P u r i n e s

in D N A

B y MIRAL DIZDAROGLU

Oxygen-derived species such as superoxide radical (O2-) and H 2 0 2 a r e

produced in mammalian cells as a result of aerobic metabolism. 1,2 Excess generation of these species in vivo by endogenous sources (e.g., oxidant enzymes, phagocytic cells) or exogenous sources (e.g., redox-cyclic drugs) can result in damage to cellular DNA. Thus, oxygen-derived species are mutagenic and may act as promoters of carcinogenesis.3,4 However, much of the toxicity of the superoxide radical and H 2 0 2 in vivo is thought to arise from their metal ion-dependent conversion into highly reactive hydroxyl radical (.OH) and iron ions appear to be the most likely catalysts of such conversion (iron-catalyzed Haber-Weiss reaction). 2 As an exoge- nous source, ionizing radiation can also cause formation df .OH among other radical species (H atoms, hydrated electrons) in cells by interaction with cellular water) In the past, radiation chemists have extensively stud- ied the reactions of free radicals with DNA and chemically characterized the resulting products) Free radicals, especially .OH, produce a large number of sugar and base products in DNA, 5 and DNA-protein cross- links in nucleoprotein in vioo and in vitro. 6-9 For an understanding of the

t I. Fridovich, Science 2119, 875 (1978). 2 B. Halliwell and J. M. C. Gutteridge, Mol. Aspects Med. 8, 89 (1985). 3 M. Larramendy, A. C. Melho-Filho, E. A. Leme Martins, and R. Meneghini, Mutat. Res.

178, 57 (1983). 4 p. A. Cerutti, Science 227, 375 (1985). 5 C. yon Sonntag, "The Chemical Basis of Radiation Biology," Taylor and Francis, London,

1987. 6 L. K. Mee and S. J. Adelstein, Proc. Natl. Acad. Sci. U.S.A. 78, 2194 (1981). 7 N. L. Oleinick, S. Chiu, N. Ramakrishnan, and L. Xue, Br. J. Cancer 55, Suppl. VIII,

135 (1987). s E. Gajewski, A. F. Fuciarelli, and M. Dizdaroglu, Int. J. Radiat. Biol. 54, 445 (1988). 9 M. Dizdaroglu, E. Gajewski, P. Reddy, and S. A. Margolis, Biochemistry 28, 3625 (1989).

Copyright © 1990 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 193 All rights of reproduction in any form reserved.

[46] G C / M S OF FREE RADICAL-INDUCED PRODUCTS 843

biological consequences of free radical-induced lesions in DNA and in nucleoprotein, it is essential to chemically characterize such lesions and measure their quantities. A number of analytical techniques has been used in the past for this purpose. This chapter describes the use of the technique of gas chromatography-mass spectrometry (GC/MS) for chemical charac- terization and quantitative measurement of free radical-induced products of pyrimidines and purines in DNA and DNA-protein cross-links in nu- cleoprotein.

Materials and Methods

Reagents. Reagents and some reference compounds used in the meth- odology described are available commercially.

Synthesis of Reference Compounds. 6-Amino-8-hydroxypurine (8-hy- droxyadenine) is synthesized from 8-bromoadenine by treatment with formic acid, and is purified by recrystallization from water. ~0 2,6-Diamino- 4-hydroxy-5-formamidopyrimidine is synthesized by treatment of 2,5,6- triamino-4-hydroxypyrimidine with formic acid, and is recrystallized from water.ll Thymine glycol is synthesized by treatment of thymine with os- mium tetroxide. 12

Hydrolysis

Prior to analysis by GC/MS, DNA and nucleoprotein must be hydro- lyzed. Acidic hydrolysis cleaves the glycosidic bonds between bases and sugar moieties in DNA and thus frees intact and modified bases. Enzymatic hydrolysis is used to hydrolyze DNA to nucleosides. In the case of DNA-protein cross-links, the simplest way for hydrolysis of nucleopro- tein appears to be the standard method of protein hydrolysis, i.e., hydroly- sis with 6 M HC1, which cleaves peptide bonds in proteins as well as glycosidic bonds in DNA to free base-amino acid cross-links, a,9 Prior to hydrolysis, DNA and nucleoprotein samples should be extensively dia- lyzed against water and subsequently lyophilized.

Acidic Hydrolysis. Of the various acids, formic acid appears to be the most suitable for hydrolysis of DNA. 13 The following procedure has been used in the studies reviewed here. An aliquot of DNA (usually 0.1-1 rag) is treated with 1 ml of formic acid (88%) in evacuated and sealed tubes at 150 ° for 30 to 40 rain. The sample is then lyophilized. This procedure of

l0 M. Dizdaroglu and D. S. Bergtoid, Anal. Bioehem. 156, 182 (1986). 11 L. F. Cavalieri and A. Bendich, J. Am. Chem. Soc. 72, 2587 (1950). 12 M. Dizdaroglu, E. Holwitt, M. P. Hagan, and W. F. Blakely, Biochem. J. 2,35, 531 (1986). 13 G. R. Wyatt and S. S. Cohen, Biochem. J. 55, 774 (1953).

844 NUCLEIC ACID CONSTITUENTS [46]

hydrolysis has been found to be optimal for quantitative removal of free radical-induced products of bases from DNA.14 Furthermore, this proce- dure does not alter the products of thymine, adenine, and guanine in DNA. 15 However, the products of free radical-damaged cytosine in DNA can be modified by deamination and/or dehydration as follows: cytosine glycol yields a mixture of 5-hydroxycytosine and 5-hydroxyuracil, the former by dehydration and the latter by deamination and dehydration. 5- Hydroxy-6-hydrocytosine, 5,6-dihydroxycytosine, and 4-amino-5-hy- droxy-2-imidazolidinon-3-ene deaminate to give 5-hydroxy-6-hydrouracil, 5,6-dihydroxyuracil, and 5-hydroxyhydantoin, respectively. These modi- fications are evidently quantitative, as indicated by studies on the release of the products as a function of the time of acidic hydrolysis as well as by the measurement of the dose-yield relationships.14

Nucleoprotein (approximately 1 mg) is hydrolyzed with 1 ml of 6 M HC1 in evacuated and sealed tubes at 120 ° for 18 hr, and then lyophilized.

Enzymatic Hydrolysis. This type of hydrolysis of DNA is discussed in detail elsewhere in this volume. 16 The following procedure has been used in the studies reviewed here. An aliquot (0.5-1 mg) of lyophilized DNA samples is incubated in 0.5 ml of 10 mM Tris-HC1 buffer, pH 8.5 (containing 2 mM MgC12) with deoxyribonuclease I (100 units), spleen exonuclease (0.01 unit), snake venom exonuclease (0.5 units), and alkaline phosphatase (10 units) at 37 ° for 24 hr. After hydrolysis, the sample is lyophilized.

The drawback of enzymatic hydrolysis is that the products of the cytosine moiety in DNA cannot readily be analyzed by gas chromatogra- phy as their nucleosides, and only one product of the thymine moiety is observed as its nucleoside. 17 On the other hand, the enzymatic hydrolysis permits the analysis of 8,5'-cyclopurine-2'-deoxynucleoside residues, which are not released from DNA by acidic hydrolysis) 7-19 In a recent work, the deamination of 2'-deoxyadenosine and its products has been observed during the course of enzymatic hydrolysis. 17 This was presum- ably due to the presence of deaminase activity in one of the enzymes used. This can be avoided by using enzymes which are free of deaminase activity.

t4 A. F. Fuciarelli, B. J. Wegher, E. Gajewski, M. Dizdaroglu, and W. F. Blakely, Radiat. Res. 119, 219 (1989).

t5 M. Dizdaroglu, Anal. Biochem. 144, 593 (1985). t6 p. F. Crain, this volume [42]. 17 M. Dizdaroglu, J. Chromatogr. 367, 357 (1986). is M. Dizdaroglu, Biochem. J. 238, 247 (1986). 19 M.-L. Dirksen, W. F. Blakely, E. Holwitt, and M. Dizdaroglu, Int. J. Radiat. Biol. 54,

195 (1988).

[46] G C / M S OF FREE RADICAL-INDUCED PRODUCTS 845

Enzymatic hydrolysis of nucleoprotein to release DNA base-amino acid cross-links for GC/MS analysis has not been reported.

Derivatization

The GC/MS technique is applicable only to compounds that are vola- tile, or can be made sufficiently volatile. Bases, nucleosides, and DNA base-amino acid cross-links are not sufficiently volatile for gas chromatog- raphy, and thus must be converted into volatile derivatives. Generally, trimethylsilylation is the most widely used mode of derivatization. The use of tert-butyldimethylsilylation has been also reported, but so far only for qualitative analysis of free radical-induced products of bases in DNA. ~5,2°,21 Trimethylsilylation is discussed in detail elsewhere in this volume. 22 The following procedure has been used for trimethylsilylation in the studies reviewed here. Lyophilized hydrolysates of DNA or nucleo- protein (0.5-1 mg) are trimethylsilylated with 0.25 ml of a mixture of bis(trimethylsilyl)trifluoroacetamide (containing 1% trimethylchlorosi- lane) and acetonitrile (1.5 : 1, by vol) at 140 ° for 30 min in polytetrafluoro- ethylene-capped vials (sealed under nitrogen). The amount of the reagents can be modified according to the amount of DNA or nucleoprotein used. After derivatization, samples are allowed to cool to room temperature, and then directly injected into the injection port of the gas chromatograph without any further treatment.

Apparatus

Any conventional GC/MS instrument equipped with a capillary inlet system and a computer work station can be used for this purpose. The present methodology utilizes fused-silica capillary columns, which are commercially available. These types of columns provide high inertness and separation efficiency, and permit measurements of high sensitivity. Columns coated with cross-linked 5% phenyl methylsilicone gum phase appear to be best for the purpose. Column length varies depending on the type of analysis. Helium (ultra high purity) is used as the carder gas. Generally, the split mode of injection is used, and the split ratio (i.e., ratio of carder gas flow through the splitter vent to flow through the column) is adjusted according to the concentration of the analyte(s) in a given mix- ture. The injection port of the gas chromatograph, the GC/MS interface, and the ion source of the mass spectrometer are kept at 250 ° . The glass

2o M. Dizdaroglu, J. Chromatogr. 295, 103 (1984). 21 M. Dizdaroglu, BioTechniques 4, 536 (1986). 22 K. H. Schram, this volume [43].

8 4 6 NUCLEIC ACID CONSTITUENTS [ 4 6 ]

O

"0

| IIM

13

f

I i i i

5 i0 15 20 TIME (min)

FIG. 1. Gas chromatogram of a DNA sample, which was y-irradiated in aqueous solution [dose, 330 Gy (joule/kg)] followed by hydrolysis with formic acid and trimethylsilylation. Column: fused-silica capillary (12 m; 0.2 mm id) coated with cross-linked 5% phenyl methyl- silicone gum phase (film thickness, 0.11/xm). Temperature program: 100 ° to 250 ° at 7°/min after 3 rain at 100 °. Peaks: I, phosphoric acid; 1, uracil; II, thymine; 2, 5,6-dihydrothymine; III, cytosine; d, 5-methylcytosine; 3, 5-hydroxy-6-hydrothymine; 4, 5-hydroxyuracil; 5, 5- hydroxy-6-hydrouracil; IIIa, cytosine; 6, 5-hydroxycytosine; 7 and 8, cis- and trans-thymine glycol; 9, 5,6-dihydroxyuracil; IV, adenine; 10, 4,6-diamino-5-formamidopyrimidine; IVa, adenine; 11, 8-hydroxyadenine; 12, 2,6-diamino-4-hydroxy-5 -formamidopyrimidine; V, gua- nine; Va, guanine; 13, 8-hydroxyguanine. Compounds represented by peaks a-g were not defined, and were also present in control samples (all compounds as their Me3 S i derivatives). (From Ref. 15 with permission.)

l iner in the in ject ion por t is filled wi th s i lanized glass wool . This pe rmi t s the h o m o g e n e o u s v a p o r i z a t i o n o f in jected samples in the inject ion por t , and avo ids p e a k tailing dur ing analys is . Mass spec t r a are r e co rded in the e lec t ron ion iza t ion (El) m o d e at 70 eV.

Gas C h r o m a t o g r a p h y - M a s s S p e c t r o m e t r y of Py r imid ine and Pur ine Products in D N A

Free B a s e s . Figure 1 i l lustrates a typica l gas c h r o m a t o g r a p h i c separa - t ion o f t r imethyls i ly l (Me3Si) de r iva t ives o f intact and modif ied bases

[46] GC/MS OF FREE RADICAL-INDUCED PRODUCTS 847

100 -

I)

73

Me3Si 0 . ~1~ C H 3 .,~" " ] ~ 0 SiMe3

M e3Si O,'~N...'~X

k 14 i 255 3 17 I?I 238 / / , 'd I I

• , kl , I, , i L, I,

m/z

FIG. 2. Mass spectrum of 5-hydroxy-6-hydrothymine-(M%Si)3.

345 /

Is9 360

100

NH SiMe3 352

N~N [ ~ N I~OSiMe3 I S iMe3 367

73 l l . . . . L . . . . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . t . . . . . . . . . . , .

0 " ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - ' - " 50 100 150 200 250 300 350 /400

ra/z

FIG. 3. Mass spectrum of 8-hydroxyadenine-(M%Si)3. (From Ref. 15 with permission.)

released by formic acid hydrolysis from y-irradiated D N A (peak identifi- cation is given in the legend). As Fig. I clearly shows, the products of all four bases are well separated from one another and from the four intact bases. Mass spectra of the trimethylsilyl (M%Si) derivatives of the modi- fied bases are dominated generally by an intense molecular ion (M .+ ion) and an intense (M - 15) + ion, 15'2° as are those of the four intact bases. 23,24 The latter ion is due to characteristic loss of a methyl radical [15 mass units (u)] from M .+ ion. 23'24 In some instances, (M - 1) ÷ ion, which results from loss of an H atom from M. + ion, also appears as an intense ion in the mass spectra. 15,2° Figures 2-4 illustrate some typical mass spectra. In its mass spectrum (Fig. 2), 5-hydroxy-6-hydrothymine-(Me3Si)3 (Mr 360) provides an intense (M - 15) + ion at m/z 345 and an intense M. + ion at m/z 360. The (M - 1) ÷ ion (m/z 359) is also produced. Loss of CO from

23 E. White, P. M. Krueger, and J. A. McCloskey, J. Org. Chem. 37, 430 (1972). 24 j. A. McCloskey, in "Basic Principles in Nucleic Acid Chemistry" (P. O. P. Ts'o, ed.),

Vol. I, p. 209. Academic Press, New York, 1974.

848 NUCLEIC ACID CONSTITUENTS [46]

i00

0 c-

-0 c

D _Q

N O SiMe3

L - ~ CH3 Me3SiO ~N--~i SiMe3

147 73 ~

/ \ . . . . . . .,.L Ik J___h, L ,J,. - - ~ /

• • i . . . . i . . . . i . . . . i ~ . ~ .

I~3~ 150 2~3~ 25~3 3 ~ m/z

FIG. 4. Mass spectrum of 5-hydroxy-5-methylhydantoin-(Me3Si)3.

331

345346 • \-/ ,

3 5 ~

(M - 15) + ion accounts for m/z 317. The m/z 271 and 255 ions result from the loss of MeaSiO. (89 u) from M. + ion and by loss of MeaSiOH (90 u) from (M - 15) + ion, respectively. Elimination of Me3SiOCN (115 u) from (M - 15) + ion, which is typical of trimethylsilylated pyrimidines and purines, 23 presumably accounts for m/z 230. Ions at m/z 73 and 147 are common fragmentation products of Me3Si derivatives and serve no diag- nostic purpose. 23 The abundant ion at m/z 130 is a companion ion of m/z 147 and is diagnostically unimportant. The mass spectrum of 8-hydroxy- adenine-(Me3Si)3 (Mr 367) is dominated by an abundant (M - 15) + ion and an abundant M. + ion at m/z 352 and 367, respectively (Fig. 3). The high abundance of these ions reflects the aromatic character of the molecule. Figure 4 illustrates the mass spectrum of 5-hydroxy-5-methylhydantoin- (Me3Si)3 (Mr 346). This compound is a major .OH-induced product of thymine and is formed in the presence of oxygen: Thus, this product is not represented in Fig. I. The (M - 15) + ion appears as the most abundant ion in the mass spectrum, whereas M .+ and (M - 1) + ions are of low intensity. Loss of CO from (M - 15) + ion accounts for m/z 303. The (M - 89) + ion is present at m/z 257. The abundant ion at m/z 216 is produced by elimination of Me3SiOCN from the (M - 15) + ion.

The separation by capillary gas chromatography of the tert-butyldi- methylsilyl derivatives of modified bases and four intact bases released from T-irradiated DNA by acidic hydrolysis has been recently reported. 21 The mass spectra of these compounds contain an intense (M - 57) + ion, which is due to typical loss of the tert-butyl radical (57 u) from M .+ ion. 21'25 In some instances, M. + and (M - 15) + ions are also observed, m

Nucleosides. The Me3Si derivatives of free radical-induced products of 2'-deoxynucleosides generally follow the same fragmentation patterns

P. F. Crain and J. A. McCloskey, Anal. Biochem. 132, 124 (1983).

[46] G C / M S O F F R E E R A D I C A L - I N D U C E D P R O D U C T S 8 4 9

100

=o -o

j3 <

0

OSiMe3

N.~N I ~ N ~ L 3~Me Me3SiHN OSiMe3 383

,o3 Me3SiOCH2 I . . . . . . . . . . t ÷H L . . . . . . . . -.~.O .~.~. H ,~2 (2s,)

H ~ ' ~ - - - J+H l.HO SiMe 3

Me3SiO ~ . 368 73 " " - . 409

I 103 147 171 [

100 150 200 250 300 350 m/z

FIG. 5. Mass spectrum of 8-hydroxy-2'-deoxyguanosine-(Me3Si)5. (From Ref. 29 with permission.)

383

x20 - - 643

409 [

410 538 628 4£2 456

139, | 1 I 1 t j , ,' I l l _ LJ~ ........ ~ ~ ....... ~ ....... ,.~ ......... ~ . . . . . . . . . -~.

400 450 500 550 600 650

as those of other nucleosides previously studied. 26,27 EI mass spectra of trimethylsilylated nucleosides are discussed in detail elsewhere in this volume. 28 As an example of free radical-induced products of nucleosides, the mass spectrum of 8-hydroxy-2'-deoxyguanosine-(MeaSi)5 (M r 643) is illustrated in Fig. 5. The M. + and (M - 15) 4 ions are present at m/z 643 and 628, respectively. The most intense ion at m/z 383 and the ion at m/z 368 represent the characterist ic (base + H) + ion [(B + 1) 4 ion] and the (base + H - Me) + ion, respectively. 26'27 The high intensity of the (B + 1) 4 ion (m/z 383) is caused by stabilization through an electron- donating substituent at the C-8 atom of the purine ring. 27,29 The ion at m/z 538 results f rom M +. ion by typical loss of Me3SiOH (90 u). 27 The ion at m/z 456 arises f rom addition of HMe3Si to the base [(B + 74) 4 ion]. Other ions are formed as illustrated in the insert in Fig. 5.

8,5 '-Cyclopurine nucleosides represent a concomitant damage to the purine base and sugar moieties of the same nucleoside. Free radical- induced formation of these compounds in DNA in vitro and in vivo has been demonstra ted recently. ~a.19.30 As expected, Me3Si derivatives of these compounds provide partly different fragmentation patterns from those of other nucleosides, t8,~9 As an example, the mass spectrum of (5'R)-8,5'-

J. A. McCloskey, A. M. Lawson, D. Tsuboyama, P. M. Krueger, and R. N. Stillwell, J. Am. Chem. Soc. 90, 4182 (1968).

27 H. Pang, K. H. Schram, D. L. Smith, S. P. Gupta, L. B. Towsend, and J. A. McCloskey, J. Org. Chem. 47, 3923 (1982). J. A. McCloskey, this volume [45].

29 M. Dizdaroglu, Biochemistry 24, 4476 (1985). 30 M. Dizdaroglu, M.-L. Dirksen, H. Jiang, and J. H. Robbins, Biochem. J. 241, 929 (1987).

850 NUCLEIC ACID CONSTITUENTS [46]

278

+Me3Si l ~,.H SiMe3 292 "'" N N

~: icaca- ] 7 3 H~i__':~-,~l . . . . . . . l . . . . J +2H I o ~ ~ oi

Me SiO ,_" . . . . . . . . J I

rz e -~,. . . . . L . . . . . . . ,L . . . . . . . I . . . . . . . . . . . . J . .~.L.J, ..,~ . ~

309

334 4G5

i ~ 2 ~ 3 ~ 4 ~ " 5 ~ 0 m/z

F]o. 6. Mass spectrum of (5'R)-8,5'-cyclo-2'-deoxyadenosine-(Me3Si)3. (From Ref. 19 with permission.)

cyclo-2'-deoxyadenosine-(Me3Si)3 (Mr 465) is illustrated in Fig. 6. The insert shows the structure of this compound and its fragmentation patterns. The (5'S)-diastereomer of this compound gives an essentially identical mass spectrum.19 Unlike other trimethylsilylated nucleosides, 8,5'-cyclo- purine nucleosides provide an abundant M. + ion in their mass spectra, 18'19 most likely due to stabilization of M .+ ion by the increased number of rings in the molecule) 1 In Fig. 6, typical (M - 15) + and (M - 15 - 90) + ions are present at m/z 450 and 360, respectively. In contrast to other trimethylsilylated nucleosides, the mass spectra of these 8,5'-cyclopurine nucleosides are characterized by the high abundance of ions containing the base plus portions of the sugar moiety. ]s,~9 One of those ions appears as the most abundant ion (m/z 309) in the mass spectrum in Fig. 6. Another difference is the absence of the (B + I) ÷ ion. Instead, the (B + Me3Si) + ion appears as an abundant peak at m/z 278. The fragmentation patterns shown in the insert in Fig. 6, which are typical of trimethylsilylated 8,5'- cyclopurine nucleosides, have been ascertained by measurement of the exact masses of the ions by high-resolution mass spectrometry.IS

DNA Base-Amino Acid Cross-Links. In the past several years, the GC/MS technique has been used extensively for the study of free radical- induced DNA base-amino acid cross-links in model systems consisting of an aqueous mixture of a DNA base and an amino acid. 8,2°,32,33 The goal of these studies was the understanding of gas chromatographic and mass spectrometric properties of cross-links and development of methodologies

31 F. W. McLafferty, "Interpretation of Mass Spectra." Univ. Sci. Books, Mill Valley, CA, 1980.

32 S. A. Margolis, B. Coxon, E. Gajewski, and M. Dizdaroglu, Biochemistry 27, 6353 (1988). 33 M. Dizdaroglu and M. G. Simic, Int. J. Radiat. Biol. 47, 63 (1985).

[ 4 6 ] G C / M S O F F R E E R A D I C A L - I N D U C E D PRODUCTS 8 5 1

@ O c"

-O c" 3

..fl

2

6

4 S 8 10 12 14 IS 18 20 Time (rnin.)

Fro. 7. Total-ion chromatogram obtained from a y-irradiated mixture of Thy and Tyr after treatment with 6 M HC1 followed by trimethylsilylation. Column: fused-silica capillary (15 m; 0.25 mm id) coated with cross-linked 5% phenyl methylsilicone gum phase (film thickness, 0.25 t~m). Temperature program: 140 ° to 270 ° at 10°/rain after 2 min at 140 °. Peaks: 1, thymine; 2, tyrosine; 3, 2-hydroxytyrosine; 4, DOPA; 5, Thy-Tyr cross-link; 6-8, dimeric products of tyrosine (all compounds as their Me3Si derivatives). (From Ref. 32 with permission.)

for identification of free radical-induced DNA-protein cross-links in nu- cleoprotein. Model systems have been exposed to ionizing radiation after N20 saturation. Under those conditions, mainly .OH was produced as a radical species and base-amino acid cross-links were formed as a result of reactions of this radical. The total-ion chromatogram of a typical model system consisting of thymine (Thy) and tyrosine (Tyr) is illustrated in Fig. 7. Under the conditions used, one Thy-Tyr cross-link was observed (peak 5). The exact structure of this compound has been elucidated by the combined use of high-performance liquid chromatography (HPLC), GC/MS, high-resolution MS, and ~H NMR and ~3C NMR spectroscopy) 2 The mass spectrum of the Me3Si derivative of the Thy-Tyr cross-link is illustrated in Fig. 8. The insert shows the structure and fragmentation patterns of the molecule. The M. + and (M - 15) + ions are present at m/z 665 and 650, respectively. The cleavage of the bond between or- and/3- carbons of the Tyr moiety accompanied by an H atom transfer gives rise to the most abundant ion at m/z 448 [(M - 218 + 1) ÷ ion]. The high abundance of this ion is most likely due to its resonance stabilization through the aromatic ring. The (M - 218) + ion (m/z 447) arising from the

852 NUCLEIC ACID CONSTITUENTS [46]

100

~J

~3

<

OSiMe3 OSlMe:=

,.,=,oX-2 4 cH 2 i :+H

,- ....... 4.- 7 ...... ~__~

i Me3StHN-- CH~CO2SlMe~ 218 548 --~

I

4 i 77

2 1 o /

e l u l IE]O 2 0 0 3 0 0 m / z 400

448

5205/48 6/5~ t . t . _ _ _ _ _ } t . . . .

5 0 0 S O 0

FIG. 8. Mass spectrum of the Me3Si derivative of Thy-Tyr cross-link.

same cleavage without the H atom transfer is also present. This cleavage, which is typical of MeaSi derivatives of amino acids, 34 also accounts for the m/z 218 ion when charge is retained on the a-carbon. Another characteristic fragmentation is the loss of .CO2SiMe 3 from M +. ion, which leads to the ion at m/z 548 [(M - 117) + ion]. Ions at m/z 520 and 622 result by typical loss of CO from m/z 548 and 650, respectively. 34 The fragmentation patterns illustrated in Fig. 8 have been ascertained by mea- surement of the exact masses of the ions by high-resolution MS. 32 The fragmentation of the Me3Si derivative of the Thy-Phe cross-link also fol- lows the same patterns giving rise to an intense (M - 218 + 1) + ion among other ions. 33 In the case of cross-links involving aliphatic amino acids, the same fragmentations occur. However , the (M - 117) + ion generally appears as one of the most prominent ions in the mass spectra, whereas the abundance of the (M - 218) + and (M - 218 + 1) + ions varies substantially depending on the aliphatic amino acid residue. 8,35

Gas Chromatography-Mass Spectrometry with Selected-Ion Monitoring

Identification at low concentrations (e.g., fcmtomole to picomole) of components of a complex mixture of organic compounds is generally carried out using GC/MS with selected-ion monitoring (SIM). 36 When

34 K. R. Leimer, R. H. Rice, and C. W. Gehrke, J. Chromatogr. 141, 355 (1977). 35 M. Dizdaroglu and E. Gajewski, Cancer Res. 49, 3463 (1989). 36 j. T. Watson, this volume [4].

[46] GC/MS OF FIIEE RADICAL-INDUCED PRODUCTS 853

2 4 6

4 6 T i m e

II

9

.jL . J , (min

10

/

10 12 )

FIG. 9. Ion-current profiles obtained during GC/MS-SIM analysis ofa DNA sample, which was treated with H202/Fe 3 ÷-NTA followed by formic acid hydrolysis and trimethylsilylation. Column as in Fig. 1 except for film thickness (0.33/zm). Temperature program: 150 ° to 250 ° at 8*/min after 2 min at 150 °. Peaks [ion monitored in the expected retention time region]: 1, 5-hydroxy-5-methylhydantoin-(Me3Si)3 (m/z 331); 2, 5-hydroxyhydantoin-(Me3Si)3 (m/z 317); 3, 5-hydroxyuracil-(Me3Si) 3 (m/z 329); 4, 5-hydroxycytosine-(Me3Si)3 (m/z 343); 5, cis-thy- mine glycol-(Me3Si)4 (m/z 259); 6, 5,6-dihydroxyuracil-(Me3Si)4 (m/z 417); 7, trans-thymine giycol-(Me3Si)4 (m/z 259); 8, 4,6-diamino-5-formamidopyrimidine-(Me3Si)3 (m/z 354); 9, 8- hydroxyadenine-(Me3Si)3 (m/z 352); 10, 2,6-diamino-4-hydroxy-5-formamidopyrimidine- (Me3Si) 4 (m/z 442); 11, 8-hydroxyguanine-(Me3Si) 4 (m/z 440).

using this technique, a mass spectrometer is set to monitor a number of typical ions of an analyte during the time interval during which the analyte elutes from the column. The analyte is then identified when the signals of the monitored ions with typical abundances all line up at its retention time. For this purpose, the knowledge of the mass spectrum and the retention time of the analyte is required. When using capillary gas chromatography, retention times can be measured with great accuracy and precision (1 in 500 or 1 in 1000; - 2 sec in 20 min, etc.), and thus play an important role in reliable identification, in addition to the simultaneous measurement of mass.

Identification of Modified Bases in DNA by GC/MS-SIM. As a typical example, Fig. 9 illustrates ion-current profiles obtained during GC/MS- SIM analysis of a trimethylsilylated hydrolysate of DNA, which was treated with H202 in aqueous solution in the presence of Fe3+-chelate of nitrilotriacetic acid (Fe3+-NTA). The H202/Fe3+-NTA system generates • OH via superoxide radical. 37 As Fig. 9 clearly shows, a large number of

37 S. Inoue and S. Kawanishi, Cancer Res. 47, 6522 (1987).

854 NUCLEIC ACID CONSTITUENTS [46]

O

H Thymine

NH2

oZ : H

Cytosine

NH2

N

Adenine

O

H 2 NH~N~--HNIH

0 0 0 0 HN OH HN OH HN H HN CH2OH HN O

O O H O O H H O 3

5-Hydroxy-6- Thymine glycol 5,6-Dihydrothymine 5-Hydrox ym et hylur a¢il 5-Hydroxy-5-methyl- hydrothymine (cis* and trans-) hydantoin

NH2 NH2 I NHHN ~ N~H~IHH N~IoH N OH O O H O H

5-Hydroxy-6- Cytosine glycol L5-Hydroxycytosine hydroc~osine

° 1 H~OH N OH N NH2

O~'~'NHI"H ] O OH O

5 Hydroxyuracil 5.6*Dihydro xy. 4-Amino-5-hydroxy- cytosine 2-imidazolidinon -3-e ne

NH2

N N N NH~HO LN I LNH 2 H H

H OHH 8-Hydroxyadenine 4,6-Diarnino-5- 8.5~Cycto - 2-' deoxyadenosine

formamidopyrimidine (5' R- and 5' .5-)

O

N NH o o

HN N HN NH-CHO H / H ~ H NH~.

H2N~N~HN~OH H2N~N~NH2 H H Guanine 8-Hydroxyguanine 2,6- Dia mino-4-hydroxy- 8,,~- Cyclo- 2'- deo xyguanosine

5-formamidopynmidine (5' R- and 5'S-)

FIG. 10. Structure of free radical-induced products of pyrimidines and purines in DNA, which were identified by the use of GC/MS technique.

products can be monitored in a single run within a short analysis time. Generally, a small amount of DNA (0.4/.~g in this case) is required for such an analysis. The amount of DNA, of course, may vary depending on the yields of the products in DNA. In Fig. 9, the current profile of only one ion of each compound is plotted in the expected retention time region. For an unequivocal identification, a number of characteristic ions of a compound are monitored in the same time interval during GC/MS-SIM analysis. Subsequently, a partial mass spectrum is obtained on the basis of the monitored ions and their relative abundances, and compared with that of authentic compound. For this purpose, the mass spectrum of the desired authentic compound should be recorded under the same tuning conditions of the mass spectrometer as are used to monitor actual samples.

Figure 10 illustrates the structure of free radical-induced products of pyrimidines and purines in DNA, which have been identified so far by the use of the GC/MS technique. 5-Hydroxy-6-hydrocytosine, 5,6-dihydroxy- cytosine, and 4-amino-5-hydroxy-2-imidazolidinon-3-ene deaminate dur-

[46] G C / M S OF FREE RADICAL-INDUCED PRODUCTS 855

ing acidic hydrolysis and are identified as their analogs derived from uracil, namely, 5-hydroxy-6-hydrouracil (peak 5 in Fig. 1), 5,6-dihydroxyuracil (peak 9 in Fig. 1 and peak 6 in Fig. 9), and 5-hydroxyhydantoin (peak 2 in Fig. 9), respectively. Cytosine glycol is also modified during acidic hydrolysis and is identified as 5-hydroxyuracil and 5-hydroxycytosine (peaks 4 and 6 in Fig. 1, and peaks 3 and 4 in Fig. 9, respectively).

Identification of DNA-Protein Cross-Links in Nucleoprotein by GC/ MS-SIM. Having obtained gas chromatographic and mass spectrometric properties of .OH-induced base-amino acid cross-links in model systems as was explained above, the GC/MS-SIM technique has been applied to identification of corresponding DNA-protein cross-links in nucleoprotein y-irradiated in aqueous solution. Calf thymus nucleohistone has been used as nucleoprotein. As a typical example, Fig. 1 1 illustrates ion-current profiles of several ions of the Me3Si derivative of the Thy-Tyr cross-link (for the mass spectrum see Fig. 8), which were obtained during GC/MS- SIM analysis of an HCl-hydrolysate of calf thymus nucleohistone. 9 The signals of the monitored ions with appropriate relative abundances are seen in Fig. 1 1A obtained with y-irradiated nucleohistone. Other DNA-protein cross-links identified in nucleohistone using GC/MS-SIM involve Thy, Cyt, Gly, Ala, Val, Leu, Ile, Thr, Lys, and Tyr. 8'35'38

Quantitative Measurements of Free Radical-Induced Products in DNA and Nucleoprotein by GC/MS-SIM. The quantity of an analyte in a com- plex mixture can often be accurately measured by GC/MS-SIM. 39 For this purpose, an internal standard is used, which is added to the samples prior to GC/MS-SIM analysis. Ideally, a stable isotope-labeled analog of the analyte is used as an internal standard as discussed elsewhere in this volume. 4° The mass spectrometer is calibrated first, using samples contain- ing known quantities of the analyte and internal standard. This is done by monitoring an intense and characteristic ion of each compound during GC/ MS-SIM analysis. The ion-current ratio of the ions monitored is plotted as a function of the ratio of the molar amounts of both compounds. The slope of such a plot is the relative molar response factor, which is used to calculate the quantity of the analyte in the mixture. Relative molar re- sponse factors depend upon experimental and instrumental conditions and should be determined in each laboratory. In the case of free radical- induced products in DNA and nucleoprotein, isotope-labeled analogs are not available. Thus, structurally similar compounds must be used as inter- nal standards. 39 The suitability of the GC/MS-SIM technique has been

E. Gajewski and M. Dizdaroglu, Biochemistry 29, 977 (1990). 39 j. T. Watson, "Introduction to Mass Spectrometry." Chap. 3. Raven, New York, 1985. 4o p. F. Crain, this volume [47].

856 NUCLEIC ACID CONSTITUENTS [46]

Ion 448 A Ion 448 B

I o n

I o n

528 Ion 528

548 Ion 548

i Ion $58 Ion $58

5 . ~ B . 8 5 . 8 Time Cmln. ) Time

i

(mln.)

FIG. 11. Ion-current profiles obtained during GC/MS-SIM analysis of a nucleohistone sample, which was hydrolyzed by HC1 and trimethylsilylated. (A) T-Irradiated in aqueous solution (dose, 300 Gy); (B) unirradiated. Column as in Fig. 1 except for internal diameter (0.32 mm) and film thickness (0.17/zm). Temperature program: 200 ° to 270 ° at 10°/min after 1 min at 200 °. (From Ref. 9 with permission.)

demonst ra ted recent ly for quanti tat ive measurement of free radical-in- duced products of pyrimidines and purines in DNA,14'19 and of free radical- induced D N A - p r o t e i n cross-l inks in nucleohistone. 8,9,35,3s

Conclusions

The GC/MS technique is well suited for analysis of modified pyrimi- dines and purines, and of D N A - p r o t e i n cross-links produced by free radical react ions in D N A and nucleoprotein. Derivat ives of these com-

[47] ANALYSIS OF 5-METHYLCYTOSINE 857

pounds possess excellent gas chromatographic properties and provide characteristic mass spectra. The high selectivity and sensitivity of the SIM technique permit the unequivocal identification of a large number of these compounds in complex hydrolysates of DNA and nucleoprotein in one single run at quantities from femtomole to picomole per injection. Their quantitative measurement is also accomplished by GC/MS-SIM using suitable internal standards. The GC/MS technique is expected to greatly contribute to the understanding of the free radical-induced damage to DNA and nucleoprotein in vitro and in vivo.

[47] Analysis of 5 -Methy lcy tos ine in D N A by Isotope Dilut ion Gas C h r o m a t o g r a p h y - M a s s Spec t rome t ry

By PAMELA F . CRAIN

Introduction

5-Methylcytosine is a minor base present in DNA from many organ- isms, and in higher eukaryotes, there is, in many cases, a general corre- spondence between hypomethylation and gene activation. 1 For cases where an accurate and unbiased direct chemical measurement of the amount of 5-methylcytosine in DNA is required, a sensitive assay has been developed based on stable isotope dilution gas chromatography-mass spectrometry (GC/MS) with selected-ion monitoring. 2-4 Nanogram amounts of DNA have been analyzed for 5-methylcytosine content from 0.2 to 1.5 tool %, and the method is suited both to the high sensitivity measurement of trace amounts of 5-methylcytosine, 2,3 and to the accurate determination of variation of 5-methylcytosine content with growth stage, 4-6 and similar studies.

The basic structure of the assay consists of the addition of isotopically labeled thymine and 5-methylcytosine to the DNA prior to hydrolysis, to

I W. Doerfler, Annu. Rev. Biochem. 52, 93 (1983). 2 p. F. Crain and J. A. McCloskey, Anal. Biochem. 132, 124 (1983). 3 j. A. McCloskey, E. M. Rachlin, C. W. Whitehead, and P. F. Crain, Nucleic Acids Res.

Syrup. Set. 20, 47 (1988). 4 p. j . Russell, J. A. Welch, E. M. Rachlin, and J. A. McCloskey, Y. Bacteriol. 169, 4393

(1987). 5 p. j . Russell, K. D. Rodland, J. E. Cutler, E. M. Rachlin, and J. A. McCloskey, "Molecular

Genetics of Filamentous Fungi," p. 321. Liss, New York, 1985. 6 p. j . Russell, K. D. Rodland, E. M. Rachlin, and J. A. McCloskey, J. Bacteriol. 169, 2902

(1987).

Copyright © 1990 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 193 All rights of reproduction in any form reserved.