Structural Studies of In Vitro Alkylation of Hemoglobin by Electrophilic

Chapter 18

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Structural Studies of In Vitro Alkylation of Hemoglobin by Electrophilic Metabolites 1

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S. Kaur , D. Hollander , R. Haas , and A. L. Burlingame 1

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Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143-0446 Air and Industrial Hygiene Laboratory, California Department of Health Services, 2151 Berkeley Way, Berkeley, CA 94704 A l k y l a t i o n of DNA by xenobiotic agents, or t h e i r e l e c t r o p h i l i c metabolites, i s b e l i e v e d to be the major initiating process that may r e s u l t u l t i m a t e l y i n carcinogenesis. The study of hemoglobin a l k y l a t e d in vivo by chemical carcinogens has p r e v i o u s l y been proposed as an i n d i c a t o r of DNA a l k y l a t i o n . X e n o b i o t i c a l l y modified p r o t e i n s , however, are not r e a d i l y amenable t o conventional methods f o r amino a c i d sequencing. Tandem mass spectrometry allows unambiguous s t r u c t u r a l e l u c i d a t i o n of chemically modified proteins. Styrene i s a widely used chemical i n the p l a s t i c s industry and its major metabolite, styrene-7,8-oxide, i s both mutagenic and carcinogenic i n rodents. Human hemoglobin was modified in vitro with styrene-7,8-oxide and digested with trypsin. T r y p t i c peptides from unmodified hemoglobin were i s o l a t e d by HPLC and their molecular weights were determined by liquid secondary ion mass spectrometry. This allowed confirmation of the known sequence of the p r o t e i n and provided a reference f o r the i d e n t i f i c a t i o n of modified peptides. High performance tandem mass spectrometry of modified peptides allowed unambiguous assignment of s p e c i f i c residues modified at the low pmol l e v e l . The e x t e r n a l l y a c c e s s i b l e h i s t i d i n e s were found t o be the dominant s i t e s f o r a l k y l a t i o n at high modif i c a t i o n l e v e l s of the p r o t e i n .

0097-6156/90/0420-0270$06.00A) © 1990 American Chemical Society Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

18. KAURETAL.

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E p i d e m i o l o g i c a l e v i d e n c e s u g g e s t s t h a t many i n d u s t r i a l chemicals are c a r c i n o g e n i c . Although the parent c o m p o u n d i t s e l f may b e i n a c t i v e , m e t a b o l i s m in vivo can produce species capable of a t t a c k i n g n u c l e o p h i l i c s i t e s i n DNA r e s u l t i n g i n c o v a l e n t a d d u c t s ( 1 - 2 ) . This process i s b e l i e v e d t o p l a y a major r o l e i n the i n i t i a t i o n of chemical carcinogenesis (1-2). E l e c t r o p h i l i c m e t a b o l i t e s f o r m e d may a l s o a t t a c k nucleophilic sites in a variety of proteins Q ) . I t has b e e n s u g g e s t e d t h a t t h e m o d i f i c a t i o n o f h e m o g l o b i n in vivo b y c a r c i n o g e n s may b e a n i n d i c a t o r o f DNA a l k y l a t i o n (4.) . The s t r u c t u r a l a s s i g n m e n t o f h e m o g l o b i n a d d u c t s may t h e r e f o r e p r o v i d e a r e a d y m e a n s o f i d e n t i f y i n g m e t a b o l i t e s o f c h e m i c a l c a r c i n o g e n s and monitoring exposure for the purposes of r i s k assessment. I n c o n t r a s t t o DNA a l k y l a t i o n , a l k y l a t i o n i n h e m o g l o b i n i s not r e p a i r e d ( £ ) . This, together with the long b i o l o g i c a l l i f e t i m e o f t h e p r o t e i n (18 w e e k s i n h u m a n s ) , a l l o w s the assessment of long-term o c c u p a t i o n a l exposure (f>) . C o v a l e n t DNA a d d u c t s f r o m t a r g e t c e l l s a r e gener a l l y d i f f i c u l t to obtain in sufficient quantity for s t r u c t u r a l s t u d i e s , p a r t i c u l a r l y s i n c e m o d i f i c a t i o n s may be removed by n o r m a l r e p a i r p r o c e s s e s . The abundance o f h e m o g l o b i n i n b l o o d (2000 n m o l p e r ml) point to the f e a s i b i l i t y of structural studies. P r e v i o u s s t u d i e s o f hemoglobin m o d i f i e d by e l e c t r o p h i l i c agents have i n c l u d e d c l e a v a g e o f the N t e r m i n a l v a l i n e adducts by r e a c t i o n w i t h p e n t a f l u o r o p h e n y l i s o t h i o c y a n a t e a n d a n a l y s i s b y GC-MS (2). This approach i s l i m i t e d to the i d e n t i f i c a t i o n of N-terminal m o d i f i c a t i o n which g e n e r a l l y represents only a s m a l l f r a c t i o n of the t o t a l covalent m o d i f i c a t i o n o f h e m o g l o b i n ( e . g . 3% f o r s t y r e n e o x i d e ) . Other approaches i n v o l v e b a s i c h y d r o l y s i s of the p r o t e i n f o l l o w e d by d e r i v a t i z a t i o n a n d G C - M S a n a l y s i s ( 8 9) o r n o n - s p e c i f i c d i g e s t i o n f o l l o w e d by FAB-MS (JJU11) . The l a t t e r approaches f a c i l i t a t e a n a l y s i s o f the entire s u i t e o f adducts formed, however, s p e c i f i c amino a c i d r e s i d u e s m o d i f i e d a r e not l i k e l y t o be r e v e a l e d . In c o n t r a s t , r e c e n t l y d e v e l o p e d tandem mass s p e c t r o m e t r i c ( t a n d e m MS) t e c h n i q u e s o f f e r t h e p o t e n t i a l f o r t h e unambiguous s t r u c t u r a l d e t e r m i n a t i o n o f the s u i t e o f modified peptides i n c l u d i n g the s p e c i f i c s i t e ( s ) of m o d i f i c a t i o n (12). Our s t r a t e g y has i n v o l v e d t h e d e t e r m i n a t i o n o f m o l e c u l a r masses o f t r y p t i c p e p t i d e s from n a t i v e human h e m o g l o b i n b y l i q u i d s e c o n d a r y i o n m a s s s p e c t r o m e t r y (LSIMS) a n d u s i n g t h e i n f o r m a t i o n as a database for the comparison of c o v a l e n t l y m o d i f i e d f

Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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hemoglobin. S i n c e m o d i f i c a t i o n s a r e d e t e c t e d as mass s h i f t s r e l a t i v e to the corresponding unmodified p e p t i d e s , the nature o f the modifying e l e c t r o p h i l e need n o t be known. Hence, t h i s a p p r o a c h o f f e r s t h e p o t e n t i a l o f i d e n t i f y i n g human e x p o s u r e t o u n k n o w n m e t a b o l i t e s o f c h e m i c a l c a r c i n o g e n s . The s p e c i f i c r e s i d u e s m o d i f i e d h a v e b e e n i d e n t i f i e d b y t a n d e m MS e x p e r i m e n t s u s i n g a n o p t i c a l l y - c o u p l e d m u l t i c h a n n e l a r r a y d e t e c t o r (13.) . S t y r e n e - 7 , 8 - o x i d e (styrene o x i d e ) , the major metabolite of the commercially important chemical s t y r e n e , was u s e d a s a m o d e l e l e c t r o p h i l e . Styrene i s w i d e l y used i n the manufacture of r e i n f o r c e d p l a s t i c s and o c c u p a t i o n a l exposure occurs mainly through i n h a l a t i o n o f t h e v a p o r ( X I ) . The m e t a b o l i t e s t y r e n e o x i d e i s m u t a g e n i c i n b o t h p r o k a r y o t i c (JJi) a n d e u k a r y o t i c t e s t systems (16.17) and c a r c i n o g e n i c i n r o d e n t s (IB.) . T h e f o r m a t i o n o f c o v a l e n t DNA a d d u c t s w i t h s t y r e n e o x i d e in vitro has been r e p o r t e d (12.), t h e r e f o r e the development o f procedures w i t h the p o t e n t i a l f o r the i d e n t i f i c a t i o n and assessment o f s t y r e n e o x i d e damage in vivo c l e a r l y need t o be explored.

Experimental

Procedures

Materials. S t a r t i n g m a t e r i a l s were o b t a i n e d as f o l l o w s : F r e s h w h o l e b l o o d was o b t a i n e d as a " s h o r t u n i t " ( I r w i n M e m o r i a l B l o o d B a n k , San F r a n c i s c o , C A ) ; h e m o g l o b i n was p r e p a r e d from washed r e d b l o o d c e l l s by c e l l l y s i s u s i n g t h r e e v o l u m e s o f s t e r i l e H2O a t 4 ° C . T h e c e l l d e b r i s w a s r e m o v e d b y c e n t r i f u g a t i o n (12 0 0 0 g , 15 m i n ) , a n d r e s i d u a l s e r u m p r o t e i n s w e r e p r e c i p i t a t e d i n 20% ( v / v ) ammonium s u l f a t e . The h e m o g l o b i n - c o n t a i n i n g s u p e r n a t a n t w a s d i a l y s e d a g a i n s t 0 . 0 1 M B i s - T r i s (pH 7 . 0 ) ; [ 8 - C ] S t y r e n e - o x i d e 2 5 m C i / m m o l , (Amersham C o r p . ) ; S t y r e n e oxide (Aldrich); TPCK-treated t r y p s i n , (Worthington); A l l s o l v e n t s w e r e HPLC g r a d e . 1 4

Instrumentation. HPLC i s o l a t i o n s w e r e p e r f o r m e d o n a B e c k m a n 421A s y s t e m u s i n g a V y d a c c o l u m n ( C - 1 8 , 4 . 6 x 250 mm). L i q u i d s e c o n d a r y i o n mass s p e c t r a (LSIMS) w e r e r e c o r d e d i n t h e p o s i t i v e i o n mode o n a K r a t o s ( M a n c h e s t e r , UK) M S - 5 0 S m a s s s p e c t r o m e t e r e q u i p p e d w i t h a 23 kG magnet a n d p o s t - a c c e l e r a t i o n d e t e c t o r . The LSIMS i o n s o u r c e has been d e s c r i b e d e l s e w h e r e ( Z Q ) . A Cs i o n b e a m o f e n e r g y 10 k e V was u s e d a s t h e p r i m a r y beam ( 2 1 ) . S p e c t r a were r e c o r d e d (300 s e c p e r d e c a d e ) w i t h a Gould ES-1000 e l e c t r o s t a t i c r e c o r d e r . T a n d e m MS e x p e r i m e n t s were p e r f o r m e d on a K r a t o s C o n c e p t IIHH ( M a n c h e s t e r , UK) f o u r s e c t o r i n s t r u m e n t o f E B E B +


18.

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geometry. T h e s a m p l e was i o n i z e d a s a b o v e i n a L S I M S i o n s o u r c e u s i n g a C s b e a m o f e n e r g y 12 k e V . O n l y t h e C i s o t o p e p e a k f o r t h e M H i o n c l u s t e r was s e l e c t e d i n MS-1 and i n t r o d u c e d i n t o a c o l l i s i o n c e l l c o n t a i n i n g helium. T h e c o l l i s i o n e n e r g y was 6 k e V a n d t h e h e l i u m p r e s s u r e i n t h e c e l l was a d j u s t e d t o o b t a i n a M H i o n a t t e n u a t i o n o f 65%. The f r a g m e n t i o n s g e n e r a t e d i n t h e c o l l i s i o n c e l l were s e p a r a t e d i n MS-2 and d e t e c t e d i n s u c c e s s i v e 4% m a s s w i n d o w s (2 s e c e x p o s u r e p e r f r a m e ) u s i n g an o p t i c a l l y - c o u p l e d 1,024 c h a n n e l a r r a y d e t e c t o r (13). S a m p l e s f o r MS a n a l y s i s w e r e t a k e n t o n e a r d r y n e s s u n d e r vacuum and r e d i s s o l v e d i n 0.1% T F A / H 2 0 . A g l y c e r o l / - t h i o g l y c e r o l / 0 . 1M HC1 ( l : l : t r a c e ) m a t r i x ( l j i l ) was a p p l i e d t o t h e s t a i n l e s s s t e e l p r o b e t i p a n d a s m a l l a l i q u o t o f t h e s a m p l e was a d d e d . The s a m p l e p r o b e t i p w a s c o o l e d d u r i n g t h e MS a n a l y s i s ( 2 2 ) . C Radioac t i v i t y measurements were o b t a i n e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g i n a Tm A n a l y t i c ( E l k G r o v e V i l l a g e , IL) instrument. +

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Hemoglobin m o d i f i c a t i o n . ( i ) H e m o g l o b i n (0.14mM) was i n c u b a t e d w i t h 25mM [ 8 - C ] s t y r e n e o x i d e ( 2 5 m C i / m m o l ) i n 8 . 5 mM B i s - T r i s (pH 7 . 0 ) , 15% ( v / v ) DMSO, 0 . 2 % ( v / v ) E t O H ( t o t a l v o l u m e 0 . 5 ml) a t 20°C i n t h e d a r k . U n r e a c t e d s t y r e n e o x i d e was r e m o v e d b y d i a l y s i s a g a i n s t 5 c h a n g e s o f 10 mM s o d i u m p h o s p h a t e , 150 mM N a C l ( 1 : 8 0 0 0 v / v , pH 7 . 4 ) . G l o b i n was i s o l a t e d a s a p r o t e i n p r e c i p i t a t e a c c o r d i n g t o t h e method o f R o s s i , F a n e l l i ££. al. (23.) . (ii) The above c o n d i t i o n s were s c a l e d - u p t o a t o t a l v o l u m e o f 20 m l u s i n g u n l a b e l e d s t y r e n e o x i d e , (iii) A r e a c t i o n m i x t u r e w i t h o u t s t y r e n e o x i d e was p r e p a r e d as a b l a n k . T P C K - t r e a t e d t r y p s i n (5u.g) w a s a d d e d t o a n a l i q u o t o f t h e g l o b i n ( 1 . 3 mg = 20 n m o l e h e m o g l o b i n ) f r o m ( i ) i n 1 5 0 \il o f 0 . 0 5 M N H 4 H C O 3 (pH 8 . 5 ) a n d t h e s o l u t i o n was i n c u b a t e d a t 37°C f o r 6 h . A f u r t h e r 5 \ig o f t h e e n z y m e was a d d e d a f t e r 2 h a n d 4 h . The t r y p t i c p e p t i d e s w e r e i s o l a t e d b y r e v e r s e d p h a s e C - 1 8 HPLC ( s o l v e n t A : 0.1% T F A , s o l v e n t B : 70% a c e t o n i t r i l e / 3 0 % H 2 O / 0 . 0 8 % 1 4

1

T F A ; s o l v e n t g r a d i e n t 0-60% B i n 60 m i n a t 1 m l m i n ' ) . F r a c t i o n s were c o l l e c t e d a t one m i n i n t e r v a l s a n d t h e r a d i o a c t i v i t y was m o n i t o r e d b y l i q u i d s c i n t i l l a t i o n counting (Fig. l b ) . An a l i q u o t o f g l o b i n from ( i i ) p r e p a r e d a s a b o v e ( 3 . 5 mg = 5 4 . 3 n m o l e h e m o g l o b i n ) w a s t a k e n u p i n 4 0 0 ^ i l o f 0 . 0 5 M NH4HCO3 (pH 8 . 5 ) a n d 40 \ig T P C K - t r e a t e d t r y p s i n was a d d e d . The s o l u t i o n was i n c u b a t e d a t 37°C f o r 6 h . A f u r t h e r 10 *ig o f t r y p s i n w a s added a f t e r 2h and 4h. The t r y p t i c p e p t i d e s were o b t a i n e d as a b o v e ( m o n i t o r i n g a t 215 nm), p o o l i n g f r a c t i o n s f r o m 5 HPLC a n a l y s e s ( F i g . l a ) . LSIMS and CID s p e c t r a o f s e l e c t e d components were r e c o r d e d . T r y p t i c


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0.0 H 0

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10

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60

70

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Time (min) F i g . 1. H P L C o f t r y p t i c d i g e s t o f human g l o b i n s (a a n d P ) f r o m h e m o g l o b i n t r e a t e d in vitro with [8- C] s t y r e n e - 7 , 8 - o x i d e (a) c h r o m a t o g r a m ; n u m b e r s r e f e r t o fractions, (b) r a d i o g r a m ; a n n o t a t i o n r e f e r s t o t r y p t i c peptide. 1 4


18.

KAURETAL.


p e p t i d e s f r o m g l o b i n (0.5mg=10 n m o l h e m o g l o b i n ) f r o m ( i i i ) , t h e b l a n k e x p e r i m e n t , were s i m i l a r l y o b t a i n e d and t h e i r m o l e c u l a r masses d e t e r m i n e d by LSIMS. The C t e r m i n a l d i p e p t i d e s were i d e n t i f i e d as t h e h e x y l e s t e r derivatives. The p r o c e d u r e f o r d e r i v a t i v e f o r m a t i o n has been d e s c r i b e d elsewhere (24). Results

and D i s c u s s i o n

H e m o g l o b i n was i s o l a t e d f r o m human e r y t h r o c y t e s and g l o b i n was p r e p a r e d b y r e m o v a l o f t h e heme m o i e t y . The g l o b i n (a a n d p c h a i n s ) was e n z y m a t i c a l l y d i g e s t e d w i t h trypsin. The r e s u l t i n g t r y p t i c p e p t i d e s were f r a c t i o n a t e d b y HPLC ( c f . F i g . l a ) a n d t h e m o l e c u l a r w e i g h t s were d e t e r m i n e d by LSIMS ( T a b l e 1 ) . The a m i n o a c i d s e q u e n c e s f o r human a a n d p g l o b i n s a r e s h o w n i n F i g u r e 2. The t r y p t i c p e p t i d e s i s o l a t e d were i d e n t i f i e d b y c o m p a r i s o n o f t h e m o l e c u l a r w e i g h t s o b t a i n e d by LSIMS w i t h t h o s e c a l c u l a t e d f o r t h e k n o w n s e q u e n c e s ( T a b l e 1, Fig. 2). M o l e c u l a r w e i g h t s were o b t a i n e d f o r a l l t h e f r a c t i o n s i n d i c a t e d i n Figure 1 w i t h the exception of f r a c t i o n s 1 and 3. Low m o l e c u l a r w e i g h t h y d r o p h i l i c p e p t i d e s a r e n o t g e n e r a l l y d e t e c t e d b y L S I M S (25.) . However, the i n t r o d u c t i o n o f h y d r o p h o b i c i t y i n such p e p t i d e s b y d e r i v a t i v e f o r m a t i o n may l e a d t o s i g n i f i c a n t i m p r o v e m e n t s i n t h e L S I M S s p e c t r u m (2Ji) . A recently reported s i n g l e step procedure for the formation of h e x y l e s t e r d e r i v a t i v e s was u s e d t o o b t a i n d e r i v a t i v e s f o r p e p t i d e s i n f r a c t i o n s 1 and 3 (24). LSIMS s p e c t r a a f t e r d e r i v a t i z a t i o n s h o w e d m o l e c u l a r i o n s M H m/z 422.2 a n d m/z 4 0 3 . 1 f o r f r a c t i o n s 1 and 3, r e s p e c t i v e l y . T h i s i d e n t i f i e d the a and P C - t e r m i n a l d i p e p t i d e s T y r A r g and T y r - H i s , r e s p e c t i v e l y ( F i g . 3, T a b l e 1 ) . LSIMS c o n f i r m e d 97% o f t h e a m i n o a c i d s e q u e n c e f o r a g l o b i n a n d 96% o f t h e s e q u e n c e f o r p g l o b i n . Tryptic peptides not detected r e p r e s e n t e d low m o l e c u l a r weight h y d r o p h i l i c peptides: G l y - H i s - G l y - L y s , cc(57-60); V a l - L y s , P(60-61); A l a - H i s - G l y - L y s , p(62-65). It is l i k e l y that t h e s e p e p t i d e s were not r e t a i n e d u n d e r t h e chromatographic conditions used. H e m o g l o b i n i s o l a t e d f r o m human e r y t h r o c y t e s was c o v a l e n t l y m o d i f i e d by i n c u b a t i n g w i t h (i) [ C-8] s t y r e n e o x i d e and ( i i ) u n l a b e l e d s t y r e n e o x i d e . Excess s t y r e n e o x i d e was r e m o v e d b y d i a l y s i s a n d t h e l e v e l o f c o v a l e n t b i n d i n g was d e t e r m i n e d b y l i q u i d s c i n t i l l a t i o n c o u n t i n g ( c a . 1% w / w ) . T r y p t i c p e p t i d e s were o b t a i n e d as above and f r a c t i o n a t e d by HPLC, m o n i t o r i n g by l i q u i d s c i n t i l l a t i o n counting. The r a d i o g r a m i n d i c a t e d a number o f p e p t i d e s c o n t a i n i n g c o v a l e n t l y b o u n d s t y r e n e oxide (Fig. l b ) . The HPLC r e t e n t i o n b e h a v i o r o f t h e r a d i o l a b e l e d c o v a l e n t a d d u c t s f r o m ( i ) was u s e d t o l o c a t e the corresponding adducts from the r e a c t i o n o f +

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LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY

a-Chain

V L S P A D K T N VK A A W G K V G A H A G E Y G A E A L E 1

8

12

17

R MF L S F P T T KT Y F P H F D L S H G S A Q V K G H G K 32

41

57

K VA D A L T N A VA H V D D M P N A L S A L S D L H A H K 62 D P V N F K LL S H C L L V T L A A H L P A E F T P A RV 93 100 V HA S L D K F L AS V S T V L T S K Y R

L

91

128

140

13-Chain V H L T P E E K S AV T A L W G K V N V D E V G G E A L G R l

9

18

L L V V Y P W T Q RF F E S F G D L S T P D A V M G N P K V 31

41

60

K AH G K K V L G AF S D G L A H L D N L K G T F A T L S E 62

67

83

L H C D K L H V D PE N F R L L G N V L V C V L A H H F G K 96

105

E F T P P V Q A A YQ K V V A G V A N A L A H K Y H 121

133

F i g . 2.

145

Amino a c i d sequences f o r human globins (a and


18. KAURETAL.


T a b l e 1. T r y p t i c p e p t i d e s f r o m human g l o b i n s (a a n d P) i d e n t i f i e d b y l i q u i d s e c o n d a r y i o n mass s p e c t r o m e t r y a f t e r C - 1 8 r e v e r s e d p h a s e HPLC f r a c t i o n a t i o n

Fraction Np,

Peptide

1

403. l

(319.1)

0(145-146)

2

461..3

(461.2)

a(8-ll)

3

422. .2*

(338.2)

a(140-141)

4

a b

a

Molecular lPn(s) (MH"? b

729. .4; 531.8 (729.4; 532.3)

a(l-7) ; o(12-16)

5

1171. .6

(1171.7)

a(l-ll)

6

952. .4

(952.5)

0(1-8)

7

1529. .4; 818.6 (1529.7; 818.4)

a(17-31) ; o(93-99)

8

1378. .4

(1378.7)

0(121-132)

9

1149. .8

(1149.7)

0(133-144)

10

1314. .9

(1314.7)

0(18-30)

11

1087. .9; 1449.9 (1087.6; 1449.8)

12

932. .8; 1421.6 (932.5; 1421.7)

a(91-99) ; 0(133-146) 0(9-17); 0(83-95)

13

1833. .8

(1833.8)

a(41-56)

14

1252. .2

(1252.7)

a(128-139)

15

1127, .0

(1126.6)

0(96-104)

16

1071, .6

(1071.6)

a(32-40)

17

1797 .5;

2529.0 (1798.0; 2529.2)

0(66-82); 0(83-104)

18

1274 .8; 2059.0 (1274.7; 2058.9)

0(31-40) ; 0(41-59)

19

1669 .5

(1669.9)

0(67-82)

20

3124, .4

(3124.6)

a(61-90)

21

2995 .9

(2996.5)

a(62-90)

22

2970 .1

(2967.6)

a(100-127)

23

1719 .8

(1720.0)

0(105-120)

MH+ o b s e r v e d (MH+ p r e d i c t e d ) . As the h e x y l e s t e r d e r i v a t i v e .


211

278


SPECTROMETRY

hemoglobin with unlabeled styrene oxide. The a p p r o p r i a t e f r a c t i o n s were c o l l e c t e d and a n a l y z e d by LSIMS t o o b t a i n m o l e c u l a r w e i g h t s o f t h e m o d i f i e d peptides. For example, modified peptide(s) with r e t e n t i o n t i m e (t ) 43-44 min i n t h e r a d i o a c t i v i t y p r o f i l e , c o r r e s p o n d e d t o f r a c t i o n 13a i n t h e HPLC chromatogram. Subsequent LSIMS o f f r a c t i o n 13a showed m o l e c u l a r i o n M H m/z 1 2 6 9 . 6 a s t h e m a j o r c o m p o n e n t (Fig. 4). T h e m i n o r c o m p o n e n t s M H m/z 1 5 6 9 . 6 a n d m/z 1833.4 are c o n s i s t e n t w i t h p e p t i d e p(133-146) c o n t a i n i n g one h y d r o x y p h e n y l e t h y l m o d i f i c a t i o n and p e p t i d e ot(4156), r e s p e c t i v e l y . P e p t i d e P(133-146) would a r i s e from i n c o m p l e t e d i g e s t i o n a t l y s i n e p(144) . P e p t i d e a n d 284 ( y ) i n d i c a t e s t h e p r e s e n c e o f a l a n i n e at t h a t p o s i t i o n . P e a k s i n t h e l o w mass r e g i o n o f C I D s p e c t r a r e p r e s e n t t h e i m m o n i u m i o n s H2N+CHR (where R i s a s i d e - c h a i n group) a r i s i n g from i n d i v i d u a l amino a c i d r e s i d u e s . The m a j o r i o n m/z 1 1 0 i n t h e immonium i o n r e g i o n o f t h e s p e c t r u m f o r f r a c t i o n 9 a r i s e s from h i s t i d i n e . The CID s p e c t r u m o f t h e m o d i f i e d p(133-144) ( f r a c t i o n 1 3 a , 20 p m o l g l o b i n ) i s s h o w n i n F i g . 6 . The R

+

+

R

1 2

1 2

x l

2


18. KAURETAL.

Structural Studies of In Vitro Alky lotion MH+ 1269

1569

i i i I i i i i n i j i I 11 i i 1833

iUU»Ul I I I I II I 1 1 1 1 1 II | || 1 I

Ai„y|llliiMlilU.^iyil 1 j-4-i-i»1111 j i n 1 1 1 1

inLlllllMiJnUll.il Jlli^iHi H.liiLb M'I

ii

1111111111111111

n

111111111

M/Z F i g . 3. L i q u i d s e c o n d a r y i o n mass s p e c t r u m o f f r a c t i o n 1 3 a s h o w i n g m o l e c u l a r i o n M H m/z 1 2 6 9 a s t h e m a j o r component, p e p t i d e P ( 1 3 3 - 1 4 4 ) . The i n s e t shows t h e m i n o r c o m p o n e n t s M H m/z 1 5 6 9 a n d m/z 1833 c o n s i s t e n t w i t h p e p t i d e P(133-146) c o n t a i n i n g one h y d r o x y p h e n y l e t h y l m o d i f i c a t i o n and p e p t i d e a ( 4 1 - 5 6 ) , respectively. P e p t i d e p(133-146) would a r i s e from incomplete cleavage at Lys p(144). +

+

422 MH+

.11 A l l

.. 1.

.1

.-

Lit

... .. A.«. . J.

403

MH+

ll.

J i l l . . . . . 1 1.1A

b

M/Z F i g . 4. L i q u i d s e c o n d a r y i o n mass s p e c t r a f o r t h e C t e r m i n a l p e p t i d e s (a) T y r - A r g , a ( 1 4 0 - 1 4 1 ) a n d (b) T y r His, p(145-146) as t h e h e x y l e s t e r d e r i v a t i v e s . Brown; Liquid Chromatography/Mass Spectrometry ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

279


90

100

no

1019 935

906

792

M/Z

708

*M

523

409

+

355

284

F i g . 5. High energy c o l l i s i o n a l l y induced d i s s o c i a t i o n mass s p e c t r u m f o r m o l e c u l a r i o n M H m/z 1149 i n f r a c t i o n 9 (see F i g . l a ) c o r r e s p o n d i n g to t r y p t i c peptide p(133-144). Significant ions i n d i c a t i n g the sequence are labeled.

1118 fl

1100

1090


M/Z +

+

Fig. 6. High energy c o l l i s i o n a l l y induced d i s s o c i a t i o n mass s p e c t r u m f o r m o l e c u l a r i o n M H m/z 1269 i n f r a c t i o n 13a (see F i g . l a ) c o r r e s p o n d i n g to t r y p t i c p e p t i d e p(133-144) c o n t a i n i n g one h y d r o x y phenylethyl modification. Fragments c o n t a i n i n g h i s t i d i n e s h o w 120 D a s h i f t c o m p a r e d t o s p e c t r u m for c o r r e s p o n d i n g u n m o d i f i e d p e p t i d e (see F i g . 5 ) . His* H i s t i d i n e m o d i f i e d w i t h C2H3OHC6H5. ** L o s s o f C2H3OHC6H5 from t h e m o l e c u l a r i o n M H .

282

UQUID CHROMATOGRAPHY/MASS SPECTROMETRY

d r a m a t i c s h i f t o f t h e h i s t i d i n e i m m o n i u m i o n m/z 110 t o m/z 2 3 0 c l e a r l y i d e n t i f i e s t h e s i t e o f m o d i f i c a t i o n b y w a y o f t h e 120 Da i n c r e a s e e x p e c t e d f o r a h y d r o x yphenylethyl moiety. T h i s i s confirmed by fragment i o n s o f t h e w, x , y a n d z s e r i e s c o n t a i n i n g h i s t i d i n e w h i c h s h o w a n a n a l o g o u s s h i f t i n 120 Da i n t h e m o d i f i e d p e p t i d e ( F i g s . 5 and 6 ) . As e x p e c t e d , i o n s o f the a, b , c and d s e r i e s (charge r e t e n t i o n at the N-terminus) do n o t show a mass s h i f t e x c e p t f o r f r a g m e n t s c o n t a i n i n g t h e h i s t i d i n e , e . g . m/z 1 0 9 5 ( a ) c o m p a r e d w i t h m/z 975 (a ) i n the unmodified peptide. This again confirms h i s t i d i n e as t h e s p e c i f i c r e s i d u e m o d i f i e d . The r e a c t i v i t y o f h i s t i d i n e toward a t t a c k by e l e c t r o p h i l e s has p r e v i o u s l y been i n d i c a t e d by the r e a c t i o n o f p o l y h i s t i d i n e w i t h s t y r e n e o x i d e (Z2.) . Reactivity of h i s t i d i n e r e s i d u e s in vivo h a s p r e v i o u s l y b e e n l x

1 1

x

d e m o n s t r a t e d b y t h e GC-MS d e t e c t i o n o f N - ( 2 h y d r o x y e t h y l ) h i s t i d i n e , as t h e m e t h y l e s t e r heptafluorobutyryl derivative, following basic h y d r o l y s i s o f g l o b i n samples i s o l a t e d from workers exposed to ethylene oxide ( £ ) . Due t o t h e l o w l e v e l o f m o d i f i c a t i o n a n d t h e c o m p l e x i t y o f t h e p e p t i d e m i x t u r e , i n some c a s e s fractions c o n t a i n i n g m o d i f i e d p e p t i d e s were p u r i f i e d by a second s t a g e o f HPLC u s i n g a p h e n y l c o l u m n . Figure 7 i l l u s t r a t e s the s i g n i f i c a n t improvement i n s i g n a l : n o i s e o b t a i n e d f o r M H 2073.9 c o r r e s p o n d i n g t o p e p t i d e (X(4156) c o n t a i n i n g t w o h y d r o x y - p h e n y l e t h y l m o d i f i c a t i o n s . The i n s e t shows t h e LSIMS s p e c t r u m a f t e r C - 1 8 f r a c t i o n a t i o n where the s i g n a l i s b a r e l y distinguished from the n o i s e . A f t e r further p u r i f i c a t i o n over a p h e n y l c o l u m n t h e s i g n a l : n o i s e i s ca. 15:1. Our s t u d y i d e n t i f i e d a number o f s i t e s o f h e m o g l o b i n m o d i f i c a t i o n w i t h styrene oxide, i n c l u d i n g the n u c l e o p h i l i c c y s t e i n e P ( 9 3 ) , a n d t h e s e r e s u l t s w i l l be p u b l i s h e d i n d e t a i l elsewhere. +

T h e a p p l i c a t i o n o f h i g h p e r f o r m a n c e t a n d e m MS t o i d e n t i f y a r a l k y l a t i o n of hemoglobin w i t h styrene oxide i l l u s t r a t e s the p o t e n t i a l o f t h i s a p p r o a c h f o r in vivo biological monitoring. At p r e s e n t , however, the s t r a t e g y o u t l i n e d i s not r e a d i l y amenable f o r m o n i t o r i n g o c c u p a t i o n a l e x p o s u r e i n humans. T h i s i s l a r g e l y due t o the high l e v e l s of unmodified peptides which co-elute d u r i n g t h e HPLC f r a c t i o n a t i o n a n d r e n d e r d i f f i c u l t the identification of modified peptides. Isolation of modi fied hemoglobin p r i o r to enzymatic d i g e s t i o n using, for e x a m p l e , i m m u n o a f f i n i t y c h r o m a t o g r a p h y may r e s u l t i n improvements i n t h i s a r e a . The c o m b i n a t i o n o f mass s p e c t r o m e t r y and immunoassay t e c h n o l o g y t h e r e f o r e offers e x c e p t i o n a l promise for the s t r u c t u r a l study of c a r c i n o g e n i n t e r a c t i o n s w i t h m a c r o m o l e c u l e s in vivo. The


18. KAURETAL.


F i g . 7. L i q u i d s e c o n d a r y i o n mass s p e c t r a f o r p e p t i d e a(41-56) c o n t a i n i n g two h y d r o x y p h e n y l e t h y l m o d i f i c a t i o n s a f t e r (a) C - 1 8 r e v e r s e d p h a s e H P L C a n d (b) C - 1 8 f o l l o w e d b y p h e n y l r e v e r s e d p h a s e H P L C .


283

284


SPECTROMETRY

m o l e c u l a r masses o f t r y p t i c p e p t i d e s from m o d i f i e d hemoglobin would allow ready i d e n t i f i c a t i o n o f covalent a d d u c t s o n t h e b a s i s o f mass s h i f t s a n d HPLC r e t e n t i o n b e h a v i o r compared w i t h u n m o d i f i e d p e p t i d e s . Subsequent h i g h p e r f o r m a n c e t a n d e m MS e x p e r i m e n t s w i t h m u l t i c h a n n e l array d e t e c t i o n would a l l o w s t r u c t u r a l assignment o f the x e n o b i o t i c e l e c t r o p h i l e and the s i t e ( s ) o f s u b s t i t u t i o n f o r samples a t t h e pmol l e v e l .

Acknowledgments We g r a t e f u l l y a c k n o w l e d g e t h e h e l p o f M r . F . C . W a l l s w i t h t h e mass s p e c t r a l a n a l y s i s . S u p p o r t e d b y NIEHS g r a n t E S 0 4 7 0 5 , N I H g r a n t RR01614 a n d NSF g r a n t D I R 8700766.

Literature

Cited

1. Miller, J. A.; Miller, E. C. In Origins of Human Cancer; Hiatt, H. H.; Watson, J. D.; Winsten J. A., Eds.; Cold Spring Harbor Laboratory, 1977; pp 605-627. 2. Grover, P. L. In Chemical Carcinogens and DNA; CRC Press: Florida, 1979. 3. Burlingame, A. L.; Straub, K.; Baillie, T. A. Mass Spectrom. Revs. 1983, 2, 331-387. 4. Osterman-Golkar, S., Ehrenberg, L., Segerback, D. and Hallstrom, L. Mutat. Res. 1976, 34, 110. 5. Calleman, C. J.; Ehrenberg, L.; Jansson, B.; Osterman-Golkar, S.; Segerback, D.; Svensson, K.; Wachtmeister, C. A. J. Environ. Pathol. Toxicol. 1978, 2, 427-442. 6. Farmer, P. B.; Bailey, E.; Gorf, S. M.; Tornqvist, M.; Osterman-Golkar, S.; Kautiainen; LewisEnright, D. P. Carcinogenesis 1986, 7, 637-640. 7. Törnqvist, M.; Mowrer, J.; Jensen, S.; Ehrenberg, L. Anal. Biochem. 1986, 154, 255-266. 8. Bailey, E.; Farmer, P. B.; Bird, I.; Lamb, J. H.; Peal, J. A. Anal. Biochem. 1986, 157, 241-248. 9. Stillwell, W. G.; Bryant, M. S.; Wishnok, J. S. Biomed. Environ. Mass Spectrom. 1987, 14, 221-223. 10. Skipper, P. L.; Obiedzinski, M. W.; Tannenbaum, S. R.; Miller, D. W.; Mitchum, R. K.; Kadlubar, F. F. Cancer Res. 1985, 45, 5122-5127. 11. Sabbioni, G.; Skipper, P. L.; Buchi, G.; Tannenbaum, S. R. Carcinogenesis 1987, 8, 819-824. 12. Hutchins, D. A.; Skipper, P. L.; Naylor, S.; Tannenbaum, S. R. Cancer Res. 1988, 48, 47564761.


18. KAURETAL. 13. 14. 15.


Burlingame, A. L. Proc. 36th Ann. Conf. Mass Spectrom. and Allied Topics, 1988, pp. 727-728. Styrene: Environmental Health C r i t e r i a 26, World Health Organization: Geneva, 1983. de Meester, C.; Poncelet, F.; Roberfroid, M.; Rondelet, J . ; Mercier, M. Mutat. Res. 1977, 5 6 , 147-152.

16. 17. 18.

Bonatti, S.; Abbondandolo, A.; C o r t i , G.; F i o r i o , R.; Mazzaccaro, A. Mutat. Res. 1978, 5 2 , 295-300. Ponomarkov, V.; Cabral, J . R. P.; Wahrendorf, J . ; Galendo, D . Cancer L e t t . 1984, 2 4 , 95-101. L i j i n s k y , W. J . N a t l . Cancer Inst. 1986, 7 7, 471476.

19. 20. 21. 22. 23. 24. 25.

Savela, K.; Hesso, A.; Hemminki, K. Chem. B i o l . Interactions 1986, 60, 235-246. F a l i c k , A. M.; Wang, G. H.; Walls, F. C. Anal. Chem. 1986, 5 8 , 1308-1311. Aberth, W.; Straub, K. M.; Burlingame, A. L. Anal. Chem. 1982, 5 4 , 2020-2034. F a l i c k , A. M.; Walls, F. C.; Laine, R. A. Anal. Biochem. 1986, 1 5 9 , 132-137. Rossi F a n e l l i , A.; Antonini, E.; Caputo, A. (1958), Biochim. Biophys. Acta 1958, 30, 608. F a l i c k , A. M.; Maltby, D . A. Anal. Biochem., i n press. Naylor, S.; F i n d e i s , A. F.; Gibson, B.W.; Williams, D . H. J . Am. Chem. Soc. 1986, 108, 6359-6363.

26. 27. 28. 29.

Sasagawa, T.; Okuyama, T.; T e l l e r , D . C. J. Chromatogr. 1982, 2 4 0 , 320-340. Tandem Mass Spectrometry, McLafferty, F. W., Ed.; Wiley: New York, 1983. Johnson, R. S.; Martin, S. A.; Biemann, K. Int. J. Mass Spectrom. Ion Proc. 1988, 86, 137-154. Hemminki, K. Carcinogenesis 1983, 4 , 1 - 3 .

RECEIVED December 5, 1989


Structural Studies of In Vitro Alkylation of Hemoglobin by Electrophilic

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