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Mass spectrometric identification and sequencing of novel neuropeptides
FION AND SEQUENCING OF NOVEL NEUROP Ph.D., Edgar Lee21 Ph.D., Jack He s Mulshine , M.D. NCI-Navy MOB, N ogy Laboratories, Ithaca, NY; 3LTP f the Health Sciences, Bethesda, M
hony Treston I, Ph.D., Phil , Alfred Yergey3, Ph.D., Center, Bethesda, MD; H, Bethesda, MD; and
is known to be an autocrin ) - the mammalian analogue of Bomb ti-BN antibody binds GRP a mall cell lung cancer (SCLC) cell in vivo ( C u t t i t t a et a l . , i n h i b i t growth of SCLC cell lines ~ l y responsive to t SCLC cell lines produce BN/GRP, a~ ody. Possible explanations become insensitive to the effects roduce autocrine hormones v i t y include i ) that these cell l i at are not bound e f f e c t i v e l y by th P antibody, or i i ) that th e interested in isolating nrelated to the BN/GRP family. We . . . . r ............ r--r......... ....................... ssical metho t i o n , p u r i f i c a t i o n , and se ,encing neur0peptides from SCLC cell lines. The classi amounts of pept ethods are therefore unsui ,cing of neur0peptides require relatively large amount ia and cell l i n e extracts .~ for the i d e n t i f i c a t i o n of peptides from sources such as spent ng modern HPLC systems wit ) i c a l l y containing only pmol to nmol amounts of a glven give peptide mino acid sequence analysi purifi ; spectrometer detectors can p o t e n t i a l l y provide o n - l ine i )eptides. MS/MS system via a direct tripl .~are u t i l i z i n g microbore HPLC columns coupled to a Sciex Sc n e u r o p e p t i d e ~ y s i s . Wi ~id introduction (DLI) atmospheric pressure ionization (API) io e f f l u e n t can be analyzed i low flow rates required for microbore HPLC (around 40 uL/min), e DLI/API/MS as low as a t t mass spectrometer, thereby providing high s e n s i t i v i t~. y Sensiti act presented at ASMS, 198 ., levels for p a r t i c u l a r neuropeptides have been obtainned (Lee e ant on the solution chemis are shownr e l a t i v e i n t e n s i t i e s of the ions in a mass spectrum a containing 2.5 n~M t r i f l u o , thereby l i m i t i n g somewhat the mobile phases which can ca be used mplex mixtures of peptides ~ic acid and up to 50% a c e t o n i t r i l e this system allows: allows a) Sepa ation of peptides on a bas • example, from conditioned tissue culture media), b) Prelimina d composition and amino ac ~heir (usually) bis-protonated molecular ion (M+2H+), and c) Bo rum of the M+2H+ ion. I uecomposition (CID ~ence information from the Collision Induced Decomposi are compiI i ng a I i brary of CID mass spectra to fTaCl faci a c i IlIIi tta~e tare a t e i1oenziT1ca~1on d ntific ano sequencln( "ng of neuropeptides wh Ict as growth-stimul atory hormonesfor SCLC. The di discovery scover of additional neuropeptides with autocrine effec CLCprol i f e r a t i on may Iead to cl eater understandi ng of the mechanismscontrollingmalignancy, and ultimatel oH bbL ies for control of SCLC. l i t devel opment of more effective therapeutic strategiq permi ,
P E P T I D E IN N O R M A L A N D D I S E A S E D ~ M U N O H I S T O C H E M I C A L L O C A L I Z A T I O N OF G A S T R I N - R E L E A S I N G IMM P a t h o l o g y , T o k a i U n i v e r s i t y S c h o o l of o HUMAN LUNG. Y u t a k a T s u t s u m i , M.D. ; D e p a r t m e n t of M e d iicine, cine
Isehara
259-11,
Japan
l o c a l i z e d in i n o r m a l a n d dis ~ s t r i n - r e l e a s i n g p e p t i d e (GRP) w a s i m m u n o h i s tt o c h e m i c a l l y Gas Formalin-fixed a n d p9aarraaffffiinn--ee m b e d d e d s p e c i m e n s w e r e s t a i n e d w i t h the e a s e d h u m a n lung. e r e i d e n t i f i e d in cells w we Isolated GRP- immunoreactive i n d iirect rect immunoperoxidase method. radually increase ~l b r o n c h i a l m u c o s a as e a r l y v as i0 w e e k s of g Qestation. GRP c e l l s gr fetal The col b o d i e s a f t e r 28 w e e k s of ges~tation. in n u m b e r a n d o f t e n f o r m e d n e u r o e p i t h e l i a l c a l i z a t i o n of c a l c i t o n i nn a n d c a l c i t o n i n g e n e - r e l a t e d p e p t i d e (CGRP) in s o m e G R P c e l l s o n f i r mmed. ed. In n o r m a l a d u l t b r o n c h i a l a n d b r o9nncchhiioo l a r m u c o s a , t h e r l a t e r f e t a l l i f e w a s confirme( w e r e o n l y a few i s o l a t e dd GRP c e l l s , w h i c h w e r e p a r t i a l l y i d e n t i c a l to c~ a l c i t o n i n cells. a n d the a b s e n c e of C G R P i The p r e s e n c e of c a l c i t o nniinn c e l l s w i t h o u t GRP i m m u n o r e a c t i v i t y A d u l t l u n g s s h o w i n g d i f f u s e or f o c a l f i b r o s i s o c c a s i o n a l l y re a d u l t l u n g w e r e of note. Dys v e a l e d f o c a l h y p e r p l a s i aa of G R P c e l l s w i t h or w i t h o u t c a l c i t o n i n i mmmmuunnoo r e a c t i v i t y . proliferation r o u n ddin( i n g p u l m o n a r y t u m o r l e t , r e a c t i v e m i c r o s c o pplc i p l a s t i c b r o n c h i o l e s surroundil b r o n c h i a l n e u r o e n d o c r i n e e c e l l s , w e r e p r e f e r e n t i a l s i t e s for s u c h h y p e r p )lasia, w h e r e a f A l l 18 p u l m o n a r y t w a s n e v e r seen. A1 A C T H c e l l s w e r e o c c a s i o nnaallll y d e m o n s t r a t e d but C G R P m o r l e t s w e r e m a i n l y com~p o s e d of G R P c e l l s , a n d a v a r y i n g n u m b e r of c a l c i t o n i n c e l l s and ACTH and GRP were detected i in s e p a r t e cells. few A C T H c e l l s w e r e r e g u l a r l y i d e n t i f i e d . Of 37 b r o n c h i a l c a r c i n o i d s , 16 (43%) c o n t a i n e d G R P i C G R P w a s r a r e l y s e e n in t u m o r l e t . munoreactive cells. T h rree e e c a r c i n o i d s w e r e l a r g e l y c o m p o s e d of G R P c e l l s a n d d e s i g n a t e d "GRPomas". S o m e of the GRP c e l l s w e r e a l s o r e a c t i v e to c a l c i t o n i n a n d CGRP, b o t h of w h i c h w e r e u s u a l l y l o c a lliizzeedd in the same t u m o r cells. A C T H was d e~tteeccttee d o n l y in six ca E i g h t e e n (40%) (40% of 45 s m a l l ce c i n o i d s , w h i l e all " G R P o m a s " c o n t a i n e d a few A C T H c e l l s . cells, a n d four fou c a r c i n o m a s s h o w e d a var'y i n g n u m b e r of G R P - i m m u n o r e a c t i v e "GRPomas" were found. C a l c i t o n i n w a s d e t e c t e d in 10 (22%) but the c o e x i s t e n c e w i t h GR ;RP in the same ca cer c e l l s w a s e x c e p t i o n a l . CGRP was almost absent from small cell carc inoma. Six of H y p e r p)lastic las n i n e A C T H - p o s i t i v e c a r c iinnoommaass r e v e a l e d GRP i m m u n o r e a c t i v i t y . i n c r e a s e of GRP c e l l s in the s u r r o u nnddiinn¢g b r o n c h i o l e s w a s s c a r c e l y n o t e d in l u n g w i t h c a r c i n o i d a n d small cell carcinoma. In c o n c l u s i o n , G R P - i m m u n o r e a c t i v e c e l l s in h u m a n l u n g e x h i b i t dy n a m i c c h a n g e s d u r i n g n o rrmma l d e v e l o p m e m t as w e l l as in f i b r o s i n g l u n g d dii s e a s e a n d in neo plastic progression. The he m e c h a n i s m of c l o s e a s s o c i a t i o n a n d d i s s o9cciiaattii o n of o t h e r regu l a t o r y p e p t i d e s in s u c h p r o c e s s e s is w o r t h i n v e s t i g a t i n g f u r t h e r .
142
hony Treston I, Ph.D., Phil , Alfred Yergey3, Ph.D., Center, Bethesda, MD; H, Bethesda, MD; and
is known to be an autocrin ) - the mammalian analogue of Bomb ti-BN antibody binds GRP a mall cell lung cancer (SCLC) cell in vivo ( C u t t i t t a et a l . , i n h i b i t growth of SCLC cell lines ~ l y responsive to t SCLC cell lines produce BN/GRP, a~ ody. Possible explanations become insensitive to the effects roduce autocrine hormones v i t y include i ) that these cell l i at are not bound e f f e c t i v e l y by th P antibody, or i i ) that th e interested in isolating nrelated to the BN/GRP family. We . . . . r ............ r--r......... ....................... ssical metho t i o n , p u r i f i c a t i o n , and se ,encing neur0peptides from SCLC cell lines. The classi amounts of pept ethods are therefore unsui ,cing of neur0peptides require relatively large amount ia and cell l i n e extracts .~ for the i d e n t i f i c a t i o n of peptides from sources such as spent ng modern HPLC systems wit ) i c a l l y containing only pmol to nmol amounts of a glven give peptide mino acid sequence analysi purifi ; spectrometer detectors can p o t e n t i a l l y provide o n - l ine i )eptides. MS/MS system via a direct tripl .~are u t i l i z i n g microbore HPLC columns coupled to a Sciex Sc n e u r o p e p t i d e ~ y s i s . Wi ~id introduction (DLI) atmospheric pressure ionization (API) io e f f l u e n t can be analyzed i low flow rates required for microbore HPLC (around 40 uL/min), e DLI/API/MS as low as a t t mass spectrometer, thereby providing high s e n s i t i v i t~. y Sensiti act presented at ASMS, 198 ., levels for p a r t i c u l a r neuropeptides have been obtainned (Lee e ant on the solution chemis are shownr e l a t i v e i n t e n s i t i e s of the ions in a mass spectrum a containing 2.5 n~M t r i f l u o , thereby l i m i t i n g somewhat the mobile phases which can ca be used mplex mixtures of peptides ~ic acid and up to 50% a c e t o n i t r i l e this system allows: allows a) Sepa ation of peptides on a bas • example, from conditioned tissue culture media), b) Prelimina d composition and amino ac ~heir (usually) bis-protonated molecular ion (M+2H+), and c) Bo rum of the M+2H+ ion. I uecomposition (CID ~ence information from the Collision Induced Decomposi are compiI i ng a I i brary of CID mass spectra to fTaCl faci a c i IlIIi tta~e tare a t e i1oenziT1ca~1on d ntific ano sequencln( "ng of neuropeptides wh Ict as growth-stimul atory hormonesfor SCLC. The di discovery scover of additional neuropeptides with autocrine effec CLCprol i f e r a t i on may Iead to cl eater understandi ng of the mechanismscontrollingmalignancy, and ultimatel oH bbL ies for control of SCLC. l i t devel opment of more effective therapeutic strategiq permi ,
P E P T I D E IN N O R M A L A N D D I S E A S E D ~ M U N O H I S T O C H E M I C A L L O C A L I Z A T I O N OF G A S T R I N - R E L E A S I N G IMM P a t h o l o g y , T o k a i U n i v e r s i t y S c h o o l of o HUMAN LUNG. Y u t a k a T s u t s u m i , M.D. ; D e p a r t m e n t of M e d iicine, cine
Isehara
259-11,
Japan
l o c a l i z e d in i n o r m a l a n d dis ~ s t r i n - r e l e a s i n g p e p t i d e (GRP) w a s i m m u n o h i s tt o c h e m i c a l l y Gas Formalin-fixed a n d p9aarraaffffiinn--ee m b e d d e d s p e c i m e n s w e r e s t a i n e d w i t h the e a s e d h u m a n lung. e r e i d e n t i f i e d in cells w we Isolated GRP- immunoreactive i n d iirect rect immunoperoxidase method. radually increase ~l b r o n c h i a l m u c o s a as e a r l y v as i0 w e e k s of g Qestation. GRP c e l l s gr fetal The col b o d i e s a f t e r 28 w e e k s of ges~tation. in n u m b e r a n d o f t e n f o r m e d n e u r o e p i t h e l i a l c a l i z a t i o n of c a l c i t o n i nn a n d c a l c i t o n i n g e n e - r e l a t e d p e p t i d e (CGRP) in s o m e G R P c e l l s o n f i r mmed. ed. In n o r m a l a d u l t b r o n c h i a l a n d b r o9nncchhiioo l a r m u c o s a , t h e r l a t e r f e t a l l i f e w a s confirme( w e r e o n l y a few i s o l a t e dd GRP c e l l s , w h i c h w e r e p a r t i a l l y i d e n t i c a l to c~ a l c i t o n i n cells. a n d the a b s e n c e of C G R P i The p r e s e n c e of c a l c i t o nniinn c e l l s w i t h o u t GRP i m m u n o r e a c t i v i t y A d u l t l u n g s s h o w i n g d i f f u s e or f o c a l f i b r o s i s o c c a s i o n a l l y re a d u l t l u n g w e r e of note. Dys v e a l e d f o c a l h y p e r p l a s i aa of G R P c e l l s w i t h or w i t h o u t c a l c i t o n i n i mmmmuunnoo r e a c t i v i t y . proliferation r o u n ddin( i n g p u l m o n a r y t u m o r l e t , r e a c t i v e m i c r o s c o pplc i p l a s t i c b r o n c h i o l e s surroundil b r o n c h i a l n e u r o e n d o c r i n e e c e l l s , w e r e p r e f e r e n t i a l s i t e s for s u c h h y p e r p )lasia, w h e r e a f A l l 18 p u l m o n a r y t w a s n e v e r seen. A1 A C T H c e l l s w e r e o c c a s i o nnaallll y d e m o n s t r a t e d but C G R P m o r l e t s w e r e m a i n l y com~p o s e d of G R P c e l l s , a n d a v a r y i n g n u m b e r of c a l c i t o n i n c e l l s and ACTH and GRP were detected i in s e p a r t e cells. few A C T H c e l l s w e r e r e g u l a r l y i d e n t i f i e d . Of 37 b r o n c h i a l c a r c i n o i d s , 16 (43%) c o n t a i n e d G R P i C G R P w a s r a r e l y s e e n in t u m o r l e t . munoreactive cells. T h rree e e c a r c i n o i d s w e r e l a r g e l y c o m p o s e d of G R P c e l l s a n d d e s i g n a t e d "GRPomas". S o m e of the GRP c e l l s w e r e a l s o r e a c t i v e to c a l c i t o n i n a n d CGRP, b o t h of w h i c h w e r e u s u a l l y l o c a lliizzeedd in the same t u m o r cells. A C T H was d e~tteeccttee d o n l y in six ca E i g h t e e n (40%) (40% of 45 s m a l l ce c i n o i d s , w h i l e all " G R P o m a s " c o n t a i n e d a few A C T H c e l l s . cells, a n d four fou c a r c i n o m a s s h o w e d a var'y i n g n u m b e r of G R P - i m m u n o r e a c t i v e "GRPomas" were found. C a l c i t o n i n w a s d e t e c t e d in 10 (22%) but the c o e x i s t e n c e w i t h GR ;RP in the same ca cer c e l l s w a s e x c e p t i o n a l . CGRP was almost absent from small cell carc inoma. Six of H y p e r p)lastic las n i n e A C T H - p o s i t i v e c a r c iinnoommaass r e v e a l e d GRP i m m u n o r e a c t i v i t y . i n c r e a s e of GRP c e l l s in the s u r r o u nnddiinn¢g b r o n c h i o l e s w a s s c a r c e l y n o t e d in l u n g w i t h c a r c i n o i d a n d small cell carcinoma. In c o n c l u s i o n , G R P - i m m u n o r e a c t i v e c e l l s in h u m a n l u n g e x h i b i t dy n a m i c c h a n g e s d u r i n g n o rrmma l d e v e l o p m e m t as w e l l as in f i b r o s i n g l u n g d dii s e a s e a n d in neo plastic progression. The he m e c h a n i s m of c l o s e a s s o c i a t i o n a n d d i s s o9cciiaattii o n of o t h e r regu l a t o r y p e p t i d e s in s u c h p r o c e s s e s is w o r t h i n v e s t i g a t i n g f u r t h e r .
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