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ELSEVIER

J o u r n a l o f C h r o m a t o g r a p h y A , 7 0 5 ( 1 9 9 5 ) 2 1 - 4 5

JOURN L OF

CHROM TOGR PHY

R e v i e w

Prote in mass spectrometry: appl icat ions to analyt ica l

b i o t e c h n o l o g y

D . N . N g u y e n * , G . W . B e c k e r , R . M . Riggin

Lil ly Research Laboratories Eli Li l ly and Company Lil ly Corporate Center Indianapolis IN 46285 US A

Abstract

T h e a d v e n t o f m a t r i x - as s i s t e d la s e r d e s o r p t i o n / i o n i z a t i o n m a s s s p e c t ro m e t r y ( M A L D I - M S ) a n d e l e c t r o s p ra y

i o n i z a t i o n m a s s s p e c t r o m e t r y ( E S I - M S ) i n th e l a s t 5 y e ar s h a s g re a t ly e n h a n c e d t h e a r e a o f p r o t e i n m a s s

s p e c t r o m e t r y . T h i s p a p e r p r e s e n t s a n o v e r v i e w o f th e a p p l i c a t io n s o f p r o t e i n m a s s s p e c t r o m e t r y i n t h e a r e a o f

a n a l y t ic a l b i o t e c h n o l o g y , p a r t i c u la r ly a s r e l a te d t o b i o p h a r m a c e u t i c a l r e s e a r c h a n d d e v e l o p m e n t . T h e s e a p p l ic a -

t i o n s i n c l u d e t h e d e t e r m i n a t i o n o f p r o t e i n m o l e c u l a r m a s s , p e p t i d e m a p p i n g , p e p t i d e s e q u e n c i n g , l i g a n d b i n d i n g ,

d e t e r m i n a t i o n o f d i s u l fi d e b o n d s , a c t i v e si t e c h a r a c t e r i z a t i o n o f e n z y m e s , p r o t e i n s e l f - a ss o c i a t i o n a n d p r o t e i n

f o l d i n g / h i g h e r o r d e r s t ru c t u r a l c h a r a c t e ri z a t i o n .

C o n t e n t s

1 . I n tr o d u ct i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2

2 . M o d e s o f m a ss s p ec t ro m e t ry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2

2 .1 . F a s t a t o m b o m b a r d m e n t m a s s s p e c tr o m e t ry ( F A B - M S ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2

2 .2 . P l a s m a d e s o r p ti o n m a s s s p e c tr o m e t ry ( P D - M S ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 4

2 .3 . M a t r ix - a ss i st e d l a se r d e s o r p ti o n / i o n i z a ti o n m a s s s p e c tr o m e t ry ( M A L D I - M S ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 5

2 .4 . E l e c tr o s p ra y i o n i za t i on m a s s s p e c tr o m e t ry ( E S I -M S ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 6

3 . P r o te i n a p p li c at io n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 8

3 .1 . M o l e c u l ar m a s s d e t e r m i n a t io n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 8

3 .2 . P e p ti d e m a p p i n g / M S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 9

3 .2 .1 . C o n f i rm a t i o n o f s e q u e n c e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 0

3 .2 .2 . P o s t- t ra n s la t i o na l m o d i fi c a ti o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1

3 .2 .3 . P r o t e in d e g r a d a ti o n p r o d u c ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3

3 .2 .4 . P r o t e i n m e t a b o li t e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4

3 .2 .5 . D i su l fi d e b o n d d e t e r m i n a ti o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 4

3 .2 .6 . L i ga n d b i nd i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5

3 .2 .7 . E n z y m e a c ti v e s it e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5

3 .2 .8 . P e p t i d e m a s s m a p s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 6

3 .3 . S e q ue n c in g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 6

3 .3 .1 . T a n d e m m a s s s p e c tr o m e t ry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 6

3 .3 .2 . P o s t- s ou r c e d e c a y M A L D I - M S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 8

* C o r r e s p o n d i n g a u t h o r .

0 0 2 1 - 9 6 7 3 / 9 5 / $ 2 9 . 0 0 © 1 9 95 E l s e v i e r S c i e n c e B . V . A l l r i g h t s r e s e r v e d

S S D 1

0 0 2 1 - 9 6 7 3 ( 9 4 ) 0 1 2 5 6 - 3

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24 D.N. Nguyen et al. / J. Chromatogr. A 705 1995) 21 -45

allows the analysis of a large number of aqueous

samples and can be used to monitor the purity of

a synthetic peptide.

Perhaps the greatest drawback of FAB and

CF-FAB is the inability to generate good signal

intensity with proteins of M r greater than about

15000, owing to involatility and thermal in-

stability of proteins, thereby limiting the general

applicability of this technique to large proteins.

The use of liquid secondary ionization mass

spectrometry (LSIMS), where the xenon beam

in FAB-MS is replaced with a stream of ions

such as Ar ÷ or Cs +, has shown some success

with proteins in the M r range 15000-20000.

However, these results are far from routine and,

in practice, FAB works best in the analysis of

proteins with molecular masses of no more than

about 5000.

2.2. P lasma desorpt ion mass spec trometry

P D - M S )

The first breakthrough in attempts to solve the

problems associated with protein mass spec-

trometry was the development of field desorp-

tion mass spectrometry [14]. In 1974, Friedman

and co-workers proposed that rapid heating of a

sample could lead to preferential desorption of

surface molecules [15]. At the same time, Mac-

farlane and co-workers discovered that when

high-energy fission fragments from 252Cf ir-

radiated thin films of arginine and cysteine,

intact molecular ions were observed [16]. Thus, a

new ionization technique, plasma desorption,

was born. Today, PD-MS is used primarily for

molecular mass determination of proteins. This

technique provides very little fragmentation, and

therefore little structural information can be

obtained. The sensitivity of this technique can be

impressive with examples of spectra obtained on

prote ins of M r about 45 000 (ovalbumin) at the

picomole level [17]. Perhaps one of the most

useful applications of this technique is its use for

peptide mapping by in situ enzymatic digestion

[18]. This procedure is normally performed with

the same nitrocellulose-bound sample as already

used for molecular mass determination. An

enzyme solution is applied and after an appro-

priate time the digestion is terminated by remov-

al of buffer by spin-drying the target. The target

containing the digested peptide fragments can

then be analyzed by PD-MS.

PD-MS has been a particularly attractive

method because of its operational simplicity and

high reliability. A schematic diagram of a PD

mass spectrometer is shown in Fig. 2. PD-MS is

based on spon taneous fission of 252Cf which

produces a pair of nuclear fragments such as

144Cs20+ and

1°6Tc22+.

These fission fragments,

with MeV energy, move in opposite directions.

One of these fragments can be oriented to hit the

sample foil and ionize the sample molecules. The

sample foil containing the adsorbed protein,

often referred to as the target, is then mounted

on a wheel assembly which can accommodate

several samples at one time. The target wheel is

positioned on the axis of the cylindrical tube

called the flight tube. The ions are accelerated by

a grid maintained at ground potential. The main

function of the fission fragment detector is to

record the time of the fission. The masses of the

ions are calculated from the time of a fission

event and the time it takes for an ion to reach

the detector. The time an ion takes to traverse

the flight tube is dependent on its mass/charge

ratio. Both detectors produce an electronic pulse

when an ion is detected.

In principle, a time-of-flight (TOF) mass spec-

trometer has no upper mass limit. However,

there are several factors which limit the range of

samples that can be analyzed by PD-MS. It is

clear that the energy density developed by the

ission Fragments

Fission Fragmen t +

Detector m ~

Na H+

0 0 0

Sample Foi l DceTrbed

Ion Detector

F i g . 2 . S c h e m a t i c d i a g r a m o f p l a s m a d e s o r p t i o n m a s s sp e c -

t r o m e t e r .

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25

f i s s ion o f 252C f i s t oo h igh a nd tha t m o s t o f t he

p r o t e i n m o l e c u l e s u n d e r g o p y r o l y s i s . T h i s p r o b -

l e m i s v e ry a p p a r e n t w h e n t h e m o l e c u la r m a s s o f

t h e p r o t e i n a p p r o a c h e s 5 0 0 0 0 . I n t h i s m a s s

r a n g e , v e r y f e w i n t a c t m o l e c u l a r i o n s a r e d e -

t e c t e d . S i n c e m o s t o f t h e p l a s m a d e s o r p t i o n i o n

s o u r c e s a r e c o u p l e d t o a T O F a n a l y z e r ( t y p i c a l

r e s o l u t i o n o f a b o u t 1 0 0 0 ) , t h e m a s s a c c u r a c y i s

f a r f r o m d e s i r a b l e . T h i s , p e r h a p s , r e s u l t s f r o m

t h e l a r g e k i n e t i c e n e r g y d i s t r i b u t i o n t h e i o n

c a r r i e s w i th i t [ 19 ]. B r oa de n ing o f t he pe a k to

t h e e x t e n t t h a t t h e i s o t o p i c p a t t e r n c a n n o t b e

r e s o l v e d i s a c o m m o n p h e n o m e n o n i n P D - M S .

T h e r e f o r e , t h e m a s s v a l u e d e r i v e d f r o m a P D -

M S i s o f t e n t h e i s o t o p i c a l l y a v e r a g e d v a l u e .

2.3. Matr ix -ass is ted laser desorpt ion / ionizat ion

m a s s s p ec tr o m et ry M A L D I - M S )

T h e d e v e l o p m e n t o f m o d e r n l a s er t ec h n o l o g y

h a s p r o v i d e d a m e a n s o f d i r e c ti n g a l ar g e a m o u n t

o f e n e r g y i n t o a s a m p l e , l e a d i n g t o d e s o r p t i o n o f

i n t a c t m o l e c u l e s r a t h e r t h a n t h e r m a l d e c o m p o s i -

t i o n . M a t r i x - a s s i s t e d l a s e r d e s o r p t i o n i o n i z a t i o n

w a s i n t r o d u c e d b y T a n a k a e t a l. [ 2 0] a n d K a r a s

a n d H i l l e n k a m p [ 21 ]. T w o r a n g e s o f l a s e r w a v e -

l e ng ths , t he f a r - in f r a r e d a nd the f a r - u l t r a v io l e t ,

a r e u s e d f o r d e s o r p t i o n o f s a m p l e m o l e c u l e s .

T h e m o s t c o m m o n l y u s e d w a v e l e n g th i s b e t w e e n

2 6 6 a n d 3 6 6 n m , w h i c h i s g e n e r a t e d f r o m a

n e o d y m i u m / y t t r i u m - a l u m i n u m - g a r n e t ( N d :

Y A G ) l a s e r . T h e r e a s o n f o r c h o o s i n g t h i s w a v e -

l e n g t h i s t h a t c o m p o u n d s c o n t a i n i n g 7 r -b o n d s ca n

b e e l e c t r o n i c a l l y e x c i t e d . W i t h r e g a r d t o m a s s

s p e c t r o m e t r y , l a s e r s o f f e r t w o i m p o r t a n t b e n e -

f i t s . F i r s t , l a se r s p r ov ide t he c a pa b i l i t y t o pu l se

f r o m a c o n t i n u o u s w a v e d o w n t o f e m t o s e c o n d

( 1 0 - 15 s ) . M o s t l a s e r m a s s s p e c t r o m e t e r s u s e

p u l s e s o f 1 0 0 n s o r l e s s t o p r e v e n t p y r o l y s i s o f

t h e p r o t e i n s . S e c o n d , l a s e r b e a m s c a n b e f o c u s e d

t o s u b m i c r o m e t e r d i a m e t e r s , a l l o w i n g t h e

o p e r a t o r t o c o n t r o l t h e l a s e r b e a m p r e c i s e l y w i t h

a n a p p r o p r i a t e m i c r o s c o p e . T h e a m o u n t o f

e n e r g y d e p o s i t e d i n t h e s a m p l e i s d e p e n d e n t o n

l a s e r i r r a d i a n c e o r i n t e n s i t y ( W c m - 2 ) , t h e p u l s e

w i d t h a n d t h e a b s o r p t i v i t y o f t h e s a m p l e . I n -

c r e a s ing the l a se r i n t e ns i t y w i l l l e a d to a n in -

c r e a s e i n d e s o r p t i o n ; h o w e v e r , f o r l a r g e p r o t e i n s

too h igh a n in t e ns i t y w i l l c e r t a in ly l e a d to e xc e s -

s i v e f r a g m e n t a t i o n .

I n M A L D I - M S , t h e s a m p l e m o l e c u l e s a r e

m i x e d w i t h a s u i t a b l e m a t r i x ( 1 : 1 0 0 0 0 r a t i o ) .

T h e c o m m o n l y u s e d m a t r i c e s a r e 3 - m e t h o x y - 4 -

hydr oxyc inna m ic a c id ( f e r u l i c a c id ) o r 3 , 5 - d i -

m e t h o x y - 4 - h y d r o x y c i n n a m i c a c i d ( s i n a p i n i c a c i d )

d i s s o l v e d i n a m i x t u r e o f a c e to n i t r i l e a n d 0 . 1 %

t r i f l u o r o a c e t i c a c i d ( T F A ) . T h e f i n a l c o n c e n t r a -

t i o n o f t h e m a t r i x i s 5 - 1 0 g /1 . T h e m a t r i x -

a n a l y t e m i x t u r e i s d e p o s i t e d o n a s i l v e r s u p p o r t

a n d a l l o w e d t o c r y s t a l l i z e b y s l o w e v a p o r a t i o n o f

t h e m a t r i x s o l v e n t s . P r o p e r s a m p l e p r e p a r a t i o n i s

e s s e n t i a l i n o b t a i n i n g a g o o d s p e c t r u m . C o - c r y s -

t a l l i z a t i on o f s a m ple a nd m a t r ix i s c r i t i c a l a nd

w i l l d e p e n d o n t h e p h y s i c a l p r o p e r t i e s o f b o t h

s a m p l e a n d m a tr ix . T h e p r e s e n c e o f T F A h a s

b e e n f o u n d t o h e l p a c h i e v e t h e c o - c r y s t a ll i z a ti o n .

A s m e n t i o n e d p r e v i o u s l y , t h e a b i l i t y t o f o c u s t h e

l a s e r b e a m p r e c i s e l y o n t h e t a r g e t a l l o w s t h e

a c c u m u l a t i o n o f s u c c e s s i v e s p e c t r a f r o m t h e

s a m e a n d / o r d i f f e r e n t a r e a s . A s i m p l e s c h e m a t i c

d i a g ra m o f a M A L D I i n s t r u m e n t i s s h o w n i n F i g .

3 . T h e i m p a c t o f a l a s e r p u l s e o n t h e t a r g e t

d e s o r b s a l a r g e n u m b e r o f io n s , w h i c h c a n c a u s e

a s a tu r a t i o n e f f e c t i n t h e d e t e c t o r . T h e h i g h

y i e l d s o f l o w - m a s s i o n s s a t u r a t e t h e d e t e c t o r ,

wh ic h r e su l t s i n l ow e r e f f i c i e nc y f o r t he h igh -

m a s s i o n s b e c a u s e t h e d e t e c t o r c a n n o t r e c o v e r

f a s t e n o u g h . T h e s o l u t i o n t o t h i s p r o b l e m i s t h e

i n s t a ll a t io n o f a n i o n d e f l e c t o r w h i c h d e f l e c t s th e

l o w - m a s s i o n s . T h e d e t e c t o r i s a l s o l o c a t e d

s li g h tl y o f f- a x is f ro m t h e m a s s s p e c t r o m e t e r . T h i s

c o m b i n a t i o n r e d u c e s a b o u t 8 0 % o f th e l o w - m a s s

i o n s . T h e l o w - m a s s i o n s d o m i n a t e t h e M r 0 - 5 0 0

Ion

eflector

lon et o~

. . . . : 0 : : : . . . . . . 1

~ e c t o r

F i g. 3 . S c h e m a t i c d i a g r a m o f M A L D I m a s s s p e c t r o m e t e r .

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26 D . N . N g u y e n e t a l . / J . C h r o m a t o g r . A 7 0 5 1 9 9 5 ) 2 1 - 4 5

range and can extend up to 1000. However,

small noise background can be seen across the

mass spectrum. The low-level noise background

is probably the result of fragmentation due to

ions colliding with the surfaces in the spectrome-

ter.

The resolution of a MALDI mass spectrome-

ter is much less than desirable, in part due to the

use of a TOF mass analyzer. An interesting, but

not surprising, fact is that the resolution for

low-mass ions is much better than that for high-

mass ions. This effect is the result of the spread

of the initial kinetic energy of the ions, and also

the existence of non-resolved adduct ions [22].

The low resolution of high-mass ions results in

uncertainty in mass determination. The uncer-

tain ty is about 10-50 u at M r 10 000 and 100-500

u for prote ins of M r 100 000.

Despite the poor resolution, sensitivity is a

major advantage of MALDI-MS. A. good signal-

to-noise spectrum can be obtained with a few

laser pulses from a single spot on the sample

target. Since successive spectra can be obtained

from a single spot, it can be concluded that this

technique is non-destructive. In practice, the

actual amount of sample consumed is in the

femtomole range. Therefore, for samples avail-

able only in limited amounts, the sample can be

recovered after the analysis. It should be pointed

out that picomole amounts of sample are nor-

mally required to form uniform matrix-analyte

crystals for the analysis.

A related technique which is still at an early

developmental stage is continuous-flow MALDI

(CF-MALDI). In CF-MALDI, the sample is

introduced to the mass spectrometer via a probe

with a liquid matrix (aqueous TFA, ethanol,

ethylene glycol, and 3-nitrobenzyl alcohol). The

laser beam is set on the opposite side of the

probe with the flight tube perpendicular to the

probe. In this present configuration, mass spec-

tra of large peptides with molecular masses

above 10 000 can be obtained [23-26].

2.4. Electrospray ioniza tion mass spectrometry

E S I - M S )

The electrospray ionization process was origi-

nally described by Dole et al. [27] in their studies

on synthetic and natural polymers of molecular

mass in excess of 100 000. However, it was not

until 10 years later that electrospray resurfaced

as a major technique in mass spectrometry. The

application of electrospray to mass spectrometry

was largely the result of two different groups

almost simultaneously. Yamashita and Fenn [28]

coupled atmospheric pressure electrospray to a

quadrupole mass spectrometer and Alexandrov

et al. [29] coupled it to a magnetic sector mass

spectrometer. A few years after the demonstra-

tion of the fundamental aspects of electrospray,

Fenn and co-workers demonstrated the ability of

electrospray to analyze high molecular mass

samples such as polyethylene glycol (Mr 17 500)

bearing a net charge of up to +23 [30]. It is this

unique feature of producing ions with multiple

charges that allows electrospray to be used in the

analysis of large proteins in a mass analyzer with

limited mass range m / z <2500 u) with an ac-

curacy of better than 0.01% [31,32].

Electrospray is produced by applying a high

electrical field to a relatively small flow of liquid

from a capillary tube. The electric field causes

the liquid surface to be highly charged and a

spray of charged liquid droplets forms at the end

of the capillary tube. The polarity of the charged

droplets can be controlled by the applied polarity

on the capillary. The mechanism by which the

molecular ions are formed from the charged

droplets is not fully understood. Iribane and

Thomson [33] proposed the field-assisted ion

evaporation model. In this model, the molecular

ion formation occurs when the field strength at

the surface of the droplet reaches a critical value

due to the evaporation of the solvent. R611gen

and co-workers proposed a different mechanism

in which the disintegration of the charged drop-

let occurs via the Rayleigh jet mechanism that

leads to very small charged droplets [34]. The

bare molecular ions are formed when complete

evaporation of the solvent from these micro-

droplets occurs. Abbas and Latham [35] showed

in a very elegant experiment that the droplets

evaporate at a constant rate until the Rayleigh

limit (1.1 x 105 elemental charges) is reached.

The droplet then abruptly loses 20-25% of its

charge and mass. This process is presumed to

continue until a bare molecular ion is formed. A

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D.N . Nguyen e t a l . / J. Chromatogr . A 705 1995) 21-4 5 2 7

p a r t i c l e o f 1 / x m r e q u i r e s a b o u t s i x t e e n d i s i n t e -

g r a t i o n s t o r e a c h t h e i o n e v a p o r a t i o n l i m i t . A t

t h i s p o i n t , m o l e c u l a r i o n p r o d u c t i o n f r o m t h e

d r o p l e t c a n t h e o r e t i c a l l y o c c u r .

I n t h e d e v e l o p m e n t o f t h e e l e c t r o s p r a y m a s s

s p e c t r o m e t e r , t r a n s m i t t i n g t h e i o n s p r o d u c e d a t

a t m o s p h e r i c p r e s s u r e i n t o t h e v a c u u m c h a m b e r

o f th e m a s s s p e c t r o m e t e r p r e s e n t e d a t r e m e n -

d o u s c h a l l e n g e . T h e m a j o r p r o b l e m i s t h e l a r g e

a m o u n t o f c o n d e n s a b l e v a p o r p r o d u c e d f r o m t h e

s p r a y n o z z l e . T h i s p r o b l e m w a s o v e r c o m e b y

a p p l y i n g a c o u n t e r f l o w i n g c u r t a i n g a s ( 2 0 - 7 0 ° C )

t o t h a t o f t h e s p r a y a n d b y n e b u l i z a ti o n . N i t r o -

g e n i s u s e d f o r c o u n t e r f l o w i n g c u r t a i n g a s . T h e

c u r t a i n g a s i s d e l i v e r e d i n t h e o p p o s i t e d i r e c t i o n

t o t h e f l o w o f s a m p l e . T h i s c u r t a i n o f d r y

n i t r o g e n s e r v e s t o e x c l u d e l a r g e d r o p l e t s a n d

p a r t i c l e s a n d t o d e c l u s t e r t h e i o n s . T h e n e b u l i z a -

t i on ga s i s u sua l ly a i r o r n i t r oge n . I t i s de l ive r e d

i n t h e s a m e d i r e c t i o n o f t h e f lo w o f s a m p l e b y

m e a n s o f a m e t a l s l e e v e w h i c h w r a p s a r o u n d t h e

c a p i l l a r y . C h a i t a n d c o - w o r k e r s s h o w e d t h a t

w h e n t h e c a p i l l a r y n o z z l e w a s o p e r a t e d a t

a r o u n d 8 5 ° C , c o u n t e r f l o w i n g g a s w a s n o t n e c e s -

s a r y [ 36 ]. H o w e v e r , t h e r m a l d e s t r u c t i o n o f l ab i l e

p e p t i d e s a n d p r o t e i n s c a n r e s u l t f r o m o p e r a t i o n

a t e l e v a t e d t e m p e r a t u r e . F i g . 4 s h o w s a

s c h e m a t i c d i a g r a m o f a n e l e c t r o s p r a y s o u r c e a n d

t h e f o r m a t i o n o f m o l e c u l a r i o n s f r o m c h a r g e d

d r o p l e t s . T h e s e m u l t i p ly c h a r g e d i o n s r e s u lt f ro m

t h e a t t a c h m e n t o f p r o t o n s a n d / o r m e t a l io n s

( N a + o r K + ) t o t h e b a s i c a n d a c id i c s i te s o n t h e

C o u n t e d l o w m 9 g a s

n

C r y o o u n l

s u r f n e e

\ \

/1L 11

u

[ I l l

O r i f i c e C r y o p u r r ~

l e c t o s p t a y

Cap i l law Ho lde r

F i g . 4 . E l e c t r o s p r a y i o n i z a t i o n s o u r c e ( a b o v e ) a n d f o r m a t i o n

o f m o l e c u l a r i o n s ( b e l o w ) .

m o l e c u l e s , r e s p e c t i v e l y . F o r e x a m p l e , t h e e -

a m i n o g r o u p o f ly s in e ca n b e p r o t o n a t e d w h e r e a s

t h e s i d e - c h a in c a r b o x y l g r o u p o f a s p a r ti c a c i d c a n

c a r r y a s o d i u m i o n a d d u c t . T h u s , t h e e l e c t r o -

s p r a y m a s s s p e c t r u m s h o w s a d i s t r i b u t i o n o f

t h e s e m u l t i p l y c h a r g e d i o n s . P r o t e i n s w i t h a

m o l e c u l a r m a s s o f m o r e t h a n 1 30 0 0 0 , s u c h a s t h e

d i m e r o f b o v i n e s e r u m a l b u m i n , h a v e b e e n

s u c c e s s f u l l y a n a l y z e d b y e l e c t r o s p r a y m a s s s p e c -

t r om e t r y [ 37 ] .

T h e d e t e r m i n a t io n o f m o l e c u l a r m a s s f r o m a n

e l e c t r o s p r a y m a s s s p e c t r u m i s s t r a i g h t f o r w a r d

g ive n the f o l lowing a s sum pt ions . F i r s t , t he a d j a -

c e n t p e a k s r e p r e s e n t s p e c i e s d i f f e r i n g b y o n l y

o n e c h a r g e . S e c o n d , t h e c h a r g e i s d u e t o p r o t o -

n a t i o n ( o r s o m e o t h e r k n o w n i o n i c s p e c i e s ) o f

t h e m o l e c u l a r i o n . A n y t w o p e a k s a r e s u f f i c i e n t

t o d e t e r m i n e t h e m o l e c u l a r m a s s . T h e r e l a t i o n -

s h i p b e t w e e n t h e m o l e c u l a r m a s s ( M r ) a n d t h e

m ul t ip ly c ha r ge d ion ( M 1 ) w i th i t s c ha r ge ( Z ~) i s

d e s c r i b e d , f o r t h e c a s e w h e r e t h e c h a r g e i s d u e

t o p r o t o n a t i o n , b y t h e e q u a t i o n

M l Z 1 = M r + 1.0079 Z 1

M 2 Z e = M r + 1 .0079 Z 2

w h e r e M 2 > M ~ . B y s o l v i n g t h e a b o v e e q u a t i o n s ,

t h e c h a r g e o f M ~ c a n b e c a l c u l a t e d u s i n g t h e

f o l lowing e qu a t io n ( Z 2 = Z ~ - 1 ):

Z l = ( M 2 - 1 . 0 0 7 9 ) / ( M z - M 1

T o d a t e , e l e c t r o s p r a y h a s b e e n m o s t w i d e l y

a p p l i e d u s i n g q u a d r u p o l e m a s s s p e c t r o m e t e r s .

O t h e r w o r k e r s h a v e d e m o n s t r a t e d t h a t e l e c t r o -

s p r a y c a n b e c o u p l e d t o o t h e r m a s s a n a l y z e r s

suc h a s m a gne t i c s e c to r [ 29 ] , t im e - o f - f l i gh t [ 38 ]

a n d F o u r i e r t r a n s f o r m i o n c y c l o t r o n r e s o n a n c e

[ 39 ] t ype s . E l e c t r ogpr a y M S i s ga in ing popu la r i t y

a m o n g p r o t e i n c h e m i s t s b e c a u s e o f it s si m p l i c it y

a n d v e r s a ti l it y . T h e f a c t t h a t e l e c t r o s p r a y M S c a n

b e c o u p l e d d i r e c t l y t o H P L C a l l o w s t h e r o u t i n e

u s e o f L C - M S . A p p l i c a ti o n s s u c h as p e p t i d e

m a p p i n g , w h i c h c o u l d t a k e h o u r s o r d a y s u s i n g

F A B - M S , c a n n o w b e d o n e w i t h E S I - M S i n a

f e w h o u r s .

U n l i k e F A B - M S , w h e r e b o t h p o s i t i v e a n d

n e g a t i v e i o n s a r e f o r m e d , o n l y p o s i t i v e o r n e g a -

t i v e i o n s a r e f o r m e d i n E S I - M S . T h e c h a r g e o f

t h e d r o p l e t s c a n b e c o n t r o l l e d b y t h e p o l a r i t y o f

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2 8

D . N . N g u y e n e t a l. / J. C h r o m a t o g r . A 7 0 5 1 9 9 5 ) 2 1 - 4 5

t he a pp l i e d e l e c t r i c a l f i e ld . I n m os t i n s t a nc e s ,

p e p t i d e s a n d p r o t e i n s w o r k w e l l i n t h e p o s i t i v e -

i o n m o d e . H o w e v e r , f o r m o l e c u l e s t h a t c o n t a i n

m a n y n e g a t i v e c h a r g e s , s u c h a s o l i g o n u c l e o ti d e s ,

t h e s a m p l e s h o u l d b e a n a l y z e d b y n e g a ti v e - i o n

ES I - M S [ 40 ] . The ne ga t ive i on ES I m a ss spe c -

t r u m i s c h a r a c t e ri z e d b y a n e n v e l o p e o f m u l t ip l y

c h a r g e d m o l e c u l a r io n s w it h th e f o r m ( M -

n i l ) - . N e g a t i v e - i o n E S I - M S g e n e r a l l y h a s l o w e r

s e n s i t i v i t y t h a n p o s i t i v e - i o n E S I - M S . T h e d e -

c r e a se i n s e ns i t i v i t y i s t he r e su l t o f subs t i t u t i on o f

s o d i u m f o r h y d r o g e n i o n s . A s t h e m o l e c u l a r

m a s s o f t h e o l i g o n u c l e o t i d e i n c re a s e s , t h e n u m -

b e r o f p h o s p h a t e - s o d i u m a d d u c t s a l so i n c re a se s .

T h i s r e s u l t s i n a b r o a d e n i n g o f t h e p e a k s a n d

l o w e r i n g o f t h e m e a s u r a b l e c u r r e n t f o r a g i v en

ion . Thus , f o r s a m ple s wh ic h a r e sub j e c t t o a lk a l i

m e t a l a t t a c h m e n t , t h e m e a s u r e d m o l e c u l a r m a s s

w i l l o f t e n b e o v e r e s t i m a t e d .

3 . P r o t e i n a p p l i c a t io n s

3 1 Molecular mass determination

T h e m o l e c u l a r m a s s o f a p r o t e i n i s a n i m -

p o r t a n t p a r a m e t e r i n t h e b i o c h e m i c a l c h a r a c t e r i -

z a t i o n o f t h a t p r o t e i n . S o d i u m d o d e c y l s u l f a t e -

p o l y a c r y la m i d e g e l e l e c tr o p h o r es i s ( S D S - P A G E )

i s a u n i v e r s a l t e c h n i q u e i n p r o t e i n m o l e c u l a r

m a s s d e t e r m i n a t i o n . H o w e v e r , t h e a c c u r a c y o f

S D S - P A G E , r a n g i n g f r o m a f e w p e r c e n t f o r a

w e l l b e h a v e d g l o b u l a r p r o t e i n t o a b o u t 3 0 % f o r

a h e a v i l y g l y c o s y l a t e d p r o t e i n , m a k e s t h e e s t i m a -

t i o n o f p r o te i n m o l e c u l a r m a s s e s e x t r e m e l y t e n u -

ous .

W i t h t h e r e c e n t a d v a n c e s i n i o n i z a t i o n t e c h -

n o l o g y d e s c r i b e d i n t h e p r e v i o u s s e c t i o n , m a s s

s p e c t r o m e t r y h a s b e c o m e a u s e f u l t o o l f o r p r o -

t e i n m o l e c u l a r m a s s d e t e r m i n a t i o n w i t h e x c e l l e n t

a c c u r a c y . T e c h n i q u e s s u c h a s M A L D I a n d E S I

a l l o w t h e d e t e r m i n a t i o n a t l o w - p i c o m o l e l e v e l s

o f p r o t e i n m o l e c u l a r m a s s e s e x c e e d i n g 1 00 0 0 0 .

T a b l e 1 s h o w s t h e t y p i c al o p e r a t i n g c h a r a c te r i s -

t i c s o f c om m e r c i a l l y a va i l a b l e i n s t r um e n t s .

I n th e d e t e r m i n a t i o n o f t h e m o l e c u l a r m a s s o f

a p r o t e i n , i t s h o u l d b e r e m e m b e r e d t h a t t h e m a s s

s p e c t r o m e t e r w i ll d e t e c t a ll i s o t o p e s o f e v e r y

e l e m e n t i n t h e m o l e c u l e . A t h i g h e n o u g h r e s -

o lu t ion , t he i on i c spe c i e s c ons i s t i ng o f d i f f e r e n t

c o m b i n a t i o n s o f i s o t o p e s w i l l b e r e s o l v e d , b u t a t

l owe r r e so lu t ion , a s ing l e sym m e t r i c a l pe a k w i l l

b e o b t a i n e d . I n E S I - M S a n d M A L D I - M S , c e n -

t r o i d in g t h e p e a k w i ll a f f o r d t h e a v e r a g e m o l e c u -

l a r m a ss o f t he p r o t e in s . F o r p r o t e in a na lys i s , i t

i s no t ne c e s sa r y t o c a r r y ou t t he a na lys i s a t h igh

r e s o l u t i o n b e c a u s e t h e m o n o i s o t o p i c i o n b e -

c o m e s u n d e t e c t a b l e , a n d e v e n t h e m o s t a b u n -

d a n t p e a k c o n t a i n s a l ar g e c o m b i n a t i o n o f is o -

t o p e s w h i c h r e q u i r e s a v e r y h i g h r e s o l u t i o n m a s s

s p e c t r o m e t e r t o a c h i e v e a c c u r a t e a n a l y s i s . A l s o ,

h i g h e r r e s o l u t i o n i s o b t a i n e d a t t h e e x p e n s e o f

sens i t iv i ty .

T h e a c c u r a c y o f E S I - M S i s a b o u t 0 . 0 1 % ,

w h i c h i s v e r y g o o d c o m p a r e d w i t h S D S - P A G E .

A l t h o u g h E S I - M S c a n m e a s u r e m o l e c u l ar m a s s e s

in e xc e s s o f 100 000 , t he a na lys i s c a n be ve r y

c o m p l i c a t e d . T h i s i s b e c a u s e E S I - M S r e q u i r e s

T a b l e 1

O p e r a t i n g c h a r a c t e ri s t ic s o f c o m m e r c i a l i n s t r u m e n t s

I o n i z a t i o n M a s s r a n g e

t e c h n i q u e

A n a l y z e r R e s o l u t i o n

T o t a l s a m p l e

r e q u i r e d f o r

d e t e r m i n a t i o n

ES I 150 000 a

M A L D I > 2 5 0 0 0 0

P l a s m a d e s o r p t i o n 4 5 0 0 0

F A B / L S I M S 5 0 0 0

1 5 0 0 0 ( L S I M S )

Q u a d r u p o l e

T i m e - o f - f l i g h tb

T i m e - o f - f l i g h t

M a g n e t i c s e c t o r

2000

1000

1000

40 000

P i c o m o l e s

P i c o m o l e s

N a n o m o l e s

N a n o m o l e s

A s s u m i n g t h e p r o t e i n s c a n b e h i g h ly p r o t o n a t e d .

b T h e o r e t i c a l l y , t h e m a s s r a n g e i s u n l i m i t e d .

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D.N . Nguyen e t a l . / J. Chromatogr . A 705 1995) 21- 45 29

t h a t t h e a d j a c e n t p e a k s b e r e s o l v e d f r o m e a c h

o t h e r , o t h e r w i s e t h e c h a r g e s t a t e c a n n o t b e

d e t e r m i n e d • T h i s i s p r o b a b l y t h e m a i n r e a s o n

w h y E S I - M S f a i l s i n a n a l y z i n g g l y c o p r o t e i n s

w h e r e h e t e r o g e n e i t y f r o m t h e c a r b o h y d r a t e m o i -

e t y c a n g i v e ri s e to a l a r g e n u m b e r o f o v e rl a p -

p i n g p e a k s . I n m a n y c a s e s M A L D I - M S c a n b e

u s e d s u c c e ss f u ll y t o d e t e r m i n e t h e m o l e c u l a r

m a s s o f g l y c o p r o t e i n s , s in c e f e w e r c h a r g e s t a te s

a r e o b s e r v e d c o m p a r e d w i t h E S I -M S .

3• • Peptide mapping~MS

P e p t i d e m a p p i n g i s a t e c h n i q u e w h e r e b y a

p r o t e i n s a m p l e i s d i g e s t e d e i t h e r e n z y m a t i c a l l y

o r c h e m i c a l l y , a n d t h e r e s u l t i n g p e p t i d e s a r e

s e p a r a t e d a n d a n a l y z e d • T h i s a p p r o a c h h a s b e e n

u s e d f o r y e a r s f o r t h e d e t e r m i n a t i o n o f p r o t e i n

p r i m a r y s e q u e n c e . O v e r l a p p i n g s e t s o f p e p t i d e

f r a g m e n t s w e r e g e n e r a t e d u s i n g d i f f e r e n t e n -

z y m e s o r c h e m i c a l a g e n t s a n d t h e p e p t i d e s w e r e

s e p a r a t e d a n d s e q u e n c e d u s i n g s t a n d a r d E d m a n

de g ra da t i on re a c t i ons • In 1981, M or r i s e t a l. [ 41 ]

u s e d p e p t i d e m a p p i n g a n d F A B - M S t o e x a m i n e

o r s c r e e n p r o t e i n d i g e s t s o r d e g r a d a t i o n p r o d -

u c t s . N o w a d a y s , p e p t i d e m a p p i n g u s e d i n c o n -

j u n c t i o n w i t h m a s s s p e c t r o m e t r y ( w e s h a l l r e f e r

t o i t h e r e a s p e p t i d e m a p p i n g / M S ) , i s u s e d i n t h e

fo l l ow i ng s i t ua t i ons : (1 ) c on f i rma t i on o f a p ro -

t e i n s e q u e n c e , e s p e c i a l l y o f p r o t e i n s p r o d u c e d

t h r o u g h r e c o m b i n a n t D N A t e c h n i q u e s , ( 2 ) d e -

t e c t i on a nd i de n t i f i c a t i on o f pos t - t r a ns l a t i ona l

mod i f i c a t i ons , (3 ) i de n t i f i c a t i on o f p ro t e i n de g ra -

d a t i o n p r o d u c t s , ( 4 ) i d e n t i f ic a t io n o f p r o t e i n

me t a bo l i t e s , (5 ) d i su l f i de bond a s s i gnme n t s , (6 )

l i g a n d b i n d i n g a n d ( 7 ) c h a r a c t e r i z a t i o n o f e n -

z ym e a c t i ve s it e s. E a c h o f t he se a p p l i c a t i ons w il l

be c ons i de re d i nd i v i dua l l y i n t he fo l l ow i ng se c -

t i o n s . A g e n e r a l p r o c e d u r e f o r p e p t i d e m a p p i n g

i s show n i n F i g . 5 . Th i s s c he me show s a l l t he

op t i ons t ha t a re a va i l a b l e ; how e ve r , i n p ra c t i c e

a l l o f t h e s e o p t i o n s a r e s e l d o m u s e d a n d t h e

u l t i m a t e g o a l o f t h e p e p t i d e m a p p i n g e x p e r i m e n t

d e t e r m i n e s t h e e x p e r i m e n t a l s t r a t e g y c h o s e n •

B r i e f l y , t h e p r o t e i n c a n b e s u b j e c t e d t o E d m a n

s e q u e n c i n g a n d c a r b o x y p e p t i d a s e d i g e s t i o n t o

d e t e r m i n e t h e N - t e r m i n a l s e q u e n c e a n d t h e C -

t e rmi na l r e s i due , r e spe c t i ve l y • The fa i l u re t o

- - E d m a n s e q u e n cin g ~ P U R I F I E D ~

C a r b o x y p e p t i d a s e l _ -

o b t a i n

2 0 - 3 0 N - t e r m i n a l I P R O T E I N I I ( A f e w C - t e r m i n a l |

r e s d u e s )

/ T / r e s id u e s , o p t i o n a l ) |

I R a d u c e / A l k y l a t e

h e p r o t e i n

I o p t i o n a l ) I

E n z y m a t i c o r c h e m i c a l o p t o n a

l e a v a g e J ] C l e a v a g e w i t h a 2 n d r e a g e n t

I ractionatev P L C

c l m a n s e q u e n c i n g

D e t e r m i n e p e p t id e m a s s e s b y

E S I -M S . M A L D I - M S o r I

F A B - M S

I D e t e r m i n e O v e r la p p i n g I

p e p t i d e m a s s e s

( t w o

e n z y m e s d i g e s t io n )

i ~ C O N F I R M T H E P R I M A R Y

S E Q U E N C E

• P r i o r k n o w l e d g e o f t h e s e q u e n c e s h e l p lu l

l o t p e p t i d e

m a p p i n g

Fig . 5 . P rocedure fo r pep t ide mapp ing .

g e n e r a t e r e s u l t s f r o m E d m a n s e q u e n c i n g s u g -

g e s t s t h a t t h e N - t e r m i n u s o f t h e p r o t e i n i s

m o d i f i e d , m o s t c o m m o n l y b y a c e ty l a t i o n o r

f o r m y l a t i o n . T h e p r o t e i n c a n t h e n b e r e d u c e d

w i t h D T T a n d a l k y l a t e d w i t h i o d o a c e t i c a c i d ,

4 - v i n y l p y r i d i n e o r a n o t h e r a l k y l a t i n g a g e n t . T h e

r e d u c e d / a l k y l a t e d p r o t e i n is t h e n s u b j e c t e d t o

c h e m i c a l o r e n z y m a t i c d i g e st i on • I n s o m e i n -

s t a n c e s i t m a y b e a p p r o p r i a t e t o o m i t t h e r e d u c -

t i o n / a l k y l a t i o n s t e p ( e . g . , f o r p r o t e i n s c o n t a i n i n g

o n l y a f e w d i s u lf i d e b o n d s o r w h e n a s s ig n i n g t h e

d i su l f i de bonds ) • The p ro t e i n c a n be d i ge s t e d

w i t h tw o d i f f e r e n t e n z y m e s i n o r d e r t o g e n e r a t e

o v e r l a p p i n g p e p t i d e f r a g m e n t s f o r m a s s s p e c t r a l

a na l ys i s • Th i s op t i on i s u se d w he n p r i o r i n fo r -

m a t i o n o n t h e p r o t e i n s e q u e n c e i s n o t a v a i l a b l e

a nd i t i s ne c e s sa ry t o a l i gn t he pe p t i de s i n t he

f i n a l s e q u e n c e • T h e d i g e s t e d f r a g m e n t s c a n b e

a n a l y z e d d i r e c t ly o r b e f r a c t i o n a t e d b y H P L C i f

t h e m i x t u r e i s t o o c o m p l e x • I f d e s i r e d , t h e

d i g e s t e d f r a g m e n t s c a n b e p u r i f ie d b y H P L C a n d

s u b j e c t e d t o E d m a n s e q u e n c i n g . T h e r e s u l t s

f r o m E d m a n s e q u e n c i n g a n d m a s s sp e c t r a l a n a l y -

s i s c a n b e c o m b i n e d t o d e d u c e t h e p r i m a r y

s t r u c t u r e o f t h e p r o t e i n . W h e n t h e N - t e r m i n u s o f

a d i ge s t e d f ra gme n t (20 re s i due s o r l e s s ) i s

b l o c k e d b y a n a c e t y l g r o u p , t h e b l o c k i n g g r o u p

c a n b e r e m o v e d b y a c y l am i n o a c y l - p ep t i d e h y d r o -

l a se [ 42 -44]• D i ge s t i on o f p ro t e i ns c a n be c a r r i e d

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30 D.N . Nguy en et al . / J . Chrornatogr. A 705 1995) 21 -4 5

Table 2

Chemical reagents and enzym es for digestion of proteins

Reagen ts/enzymes Site of cleavage

1. Trypsin

2. S. aureus V8, pH 8

3. S. aureus V8, pH 4

4. Chymotrypsin

5. Endo protease Lys-C

6. Endo protease Asp-N

7. Clostripain

8. Pepsin

9. Thermolysin

10. CNBr

11. Hydroxylamine

12. Di lute acetic acid

13. Cysteine cyanylation

14. NCS

15. Iodosobenzoicacid

C-terminus

C-terminus

C-terminus

C-terminus

C-terminus

N-terminus

C-terminus

C-terminus

N-terminus

of Arg and Lys

of Glu and Asp

of Glu

of Phe, Tyr, Trp, Leu and M et

of Lys

of Asp

of Arg

of Ph e, Met , Leu and Trp

of Leu, l ie, V al, Ph e, Met and Ala

C-terminus of Met

Asn-Gly bond

Asp-Pro bond

N-terminus of Cys

C-terminus of Trp

C-terminus of Trp

o u t c h e m i c a l l y o r e n z y m a t i c a l l y . T a b l e 2 li st s t h e

c o m m o n c h e m i c a l re a g e n t s a n d e n z y m e s t h a t a re

u s e d i n p e p t i d e m a p p i n g .

A s m e n t i o n e d p r e v i o u s l y , c e r t a i n i o n s u p p r e s -

s i o n e f fe c t s w e r e o b s e r v e d i n F A B - M S w i t h

h y d r o p h i l i c p e p t i d e s . T h i s e f f e c t i s v i r t u a l l y n o n -

e x i s t e n t w h e n p e p t i d e m a p p i n g i s c a r r i e d o u t

w i t h P D - M S o r E S I - M S . T h e c a p a b i li t y o f c o u -

p l i n g e l e c tr o s p r a y t o H P L C a l lo w s t h e a n a l y s is o f

t h e d i g e s t i o n f r a g m e n t s w i t h o u t a n y p u r i f i c a t i o n

o r f r a c t i o n a t i o n . W i t h E S I - M S , f u ll s e q u e n c i n g

i n f o r m a t i o n i s o f t e n o b t a i n e d b y p e r f o r m i n g

c o l l is i o n - i n d u c e d d i s s o c ia t i o n t a n d e m m a s s s p e c -

t r o m e t r y ( M S - M S ) . T h e a p p l ic a ti o n o f t h e M S -

M S t e c h n i q u e i n s e q u e n c i n g w i l l b e d i s c u s s e d

l a t e r .

3 . 2 . 1 . C o n f i r m a t i o n o f s e q u e n c e

W i t h t h e p r o l i f e r a ti o n o f p r o t e i n s b e i n g p r o -

d u c e d b y r e c o m b i n a n t D N A t e c h n iq u e s u si n g a

v a r i e t y o f e x p r e s s i o n s y s t e m s , i t i s p r u d e n t t o

c o n f i r m t h a t t h e p r o t e i n s e q u e n c e o b t a i n e d i s i n

a g r e e m e n t w i t h t h a t p r e d i c t e d f r o m t h e D N A

s e q u e n c e . P e p t i d e m a p p i n g / M S i s a n i d ea l p r o -

c e d u r e f o r p e r f o r m i n g t h i s c o n f i r m a t i o n o f s tr u c -

t u r e i n a ra p i d f a s h i o n w i t h m i n i m a l c o n s u m p t i o n

o f p r o t e i n s a m p l e . T h e m o l e c u l a r m a s s e s o f

t r y p t i c p e p t i d e s f r o m t h e p r e d i c t e d s e q u e n c e a r e

c o m p a r e d w i t h t h e m o l e c u l a r m a s s e s a c t u a l l y

d e t e r m i n e d f r o m th e p e p t id e m a p p i n g / M S e x -

p e r i m e n t . T h i s s t r a t e g y w a s f i rs t s u g g e s t e d i n

1 98 4 b y G i b s o n a n d B i e m a n n [4 5], w h o u s e d i t

t o c o n f i r m a n d c o r r e c t r e g i o n s f r o m t h e a m i n o

a c i d s e q u e n c e s o f t h r e e l a rg e p r o t e i n s ,

g l u t a m i n y l - a n d g l y c y l - t R N A sy n t h e t a s e f r o m

E s c h e r i c h i a c o l i a n d m e t h i o n y l - t R N A s y n th e t a s e

f r o m y e a s t . S i n c e t h a t t i m e t h e r e h a v e b e e n

s e v e ra l e x a m p l e s o f th i s sa m e a p p r o a c h a p p l i e d

t o o t h e r p r o t e i n s [ 4 6 - 4 9 ] . I n t h e p a s t , p e p t i d e

m a p p i n g w a s a l a b o r i o u s p r o c e s s w h i c h i n c l u d e d

o p t i m i z a t i o n o f t h e H P L C c o n d i t i o n s , fr a c t i o n a -

t i o n o f a l l t h e d i g e s t e d p e p t i d e f r a g m e n t s a n d

a n a l y s i s o f a l l f r a c t i o n s b y e i t h e r a m i n o a c i d

a n a l y s i s , p e p t i d e s e q u e n c i n g o r m a s s s p e c -

t r o m e t r y o r a l l t h r e e . W i t h m o d e r n t e c h n o l o g i e s

s u ch as E S I - M S a n d M A L D I - M S , p e p t i d e m a p -

p i n g / M S o f a d i g e s t e d p e p t i d e m i x t u r e c a n b e

p e r f o r m e d i n a m u c h s h o r t e r t i m e .

M A L D I - M S h a s p r o v e n u s e fu l f o r p e p t i d e

m a p p i n g / M S a n d t h e r e a r e s ev e r al a d v a n t a g e s i n

u s in g th e s e t e ch n i q u e s. S i nc e M A L D I - M S c a n

t o l e r a t e t h e p r e s e n c e o f b u f f e r s al ts a n d o t h e r

i m p u r i t i e s, a m i x t u r e o f p e p t i d e s o b t a i n e d f r o m ,

f o r e x a m p l e , a n e n z y m a t i c d i g e s t c a n b e a n a -

l y z e d w i t h o u t p r i o r p u r i f i c a ti o n . T h i s t e c h n i q u e

a l lo w s a m a s s s p e c t r u m c o n t a i n i n g i o n m a s s e s f o r

a l l p e p t i d e f r a g m e n t s t o b e o b t a i n e d i n o n l y a

f e w m i n u t e s a f t e r c o m p l e t i o n o f t h e d i g e s t .

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D . N . N g u y e n e t a l. / J . C h r o m a t o g r . A 7 0 5 1 9 9 5 ) 2 1 - 4 5 31

D i s a d v a n t a g e s o f t h i s t e c h n i q u e a r e g e n e ra l l y

r e l a t e d t o t h e m a t r i x i n w h i c h t h e s a m p l e i s

a n a l y z e d . T h e a n a l y t e s m u s t b e u n i f o r m l y d i s -

t r i b u t e d t h r o u g h o u t t h e m a t r i x c r y s t a l s t o e n s u r e

t h a t a l l p e p t i d e s c a n b e d e s o r b e d . T h e c h o i c e o f

m a t r i x i s c r it i ca l . E a c h o f th e c o m m o n m a t r ic e s

d i s c r i m i n a t e s a g a i n s t m i x t u r e c o m p o n e n t s i n

w a y s th a t a r e s a m p l e a n d m a t r ix s p e c i fi c , t h e r e b y

n e c e s s i t a t i n g t h a t d a t a b e a c q u i r e d u s i n g m o r e

t h a n o n e m a t r i x . B a c k g r o u n d s i g n a l s a r e a p r o b -

l e m w i t h a n y m a t r i x a n d w i t h s o m e t h e s i g n a l s

a r e p r e s e n t u p t o M r 1 00 0. M a n y p e p t i d e m i x -

t u r e s r e s u l ti n g f r o m f r a g m e n t a t i o n o f a p r o t e i n

c o n t a i n p e p t i d e s w i t h m o l e c u l a r m a s s e s w e l l

b e l o w 1 0 0 0 . T h e s e p r o b l e m s a r e b e i n g s l o w l y

o v e r c o m e b y t h e d e v e l o p m e n t o f n e w m a t r i c e s

a n d n e w e r p r o c e d u r e s f o r s a m p l e p r e p a r a t i o n .

S tu l t s e t a l . [ 50 ] e va lua t e d the e f f e c t s o f c a r bohy-

d r a t e- c o n t ai n i n g m a t r ic e s f o r M A L D I - M S a n al y -

s i s o f i n s i t u d i g e s t s o f p r o t e i n s f r o m t w o - d i m e n -

s i o n a l g e l s a n d f o u n d f u c o s e - 2 , 5 - d i h y d r o x y b e n -

z o i c a c i d ( D H B ) t o b e t h e b e s t . S e v e r a l c r y s -

t a l l i z a t i o n m e t h o d s f o r M A L D I - M S h a v e a l s o

b e e n r e p o r t e d , w h i c h i n c l u d e v a c u u m c r y s t a l l i z a -

t i on [ 51 ] a nd s t r e s se d m a t r ix c r ys t a l l i z a t i on

[52,53] .

E S I - M S h a s o n e m a j o r a d v a n t a g e o v e r

M A L D I - M S , n a m e l y t h e a b il it y t o c o u p le a n

H P L C s y s t e m d i r e c t l y t o t h e m a s s s p e c t r o m e t e r

a n d p e r f o r m L C - M S . A p p l i c a ti o n o f E S I - M S in

p e p t i d e m a p p i n g / M S o f f e r s a r a p i d m e a n s t o

c o n f i r m t h e p r i m a r y s e q u e n c e o f p r o te i n s . I n

g e n e r a l , t h e d i g e s t i o n m i x t u r e i s s e p a r a t e d b y

R P - H P L C . T h e o u t p u t f r o m H P L C i s s p li t; a

s m a l l a m o u n t o f e f f l u e n t g o e s t o t h e m a s s s p e c -

t r o m e t e r a n d t h e r e s t g o e s t o a f r a c t i o n c o l l e c t o r

o r w a s t e . U s i n g t h i s s e t - u p t h e m a s s o f a p a r -

t i c u l a r H P L C p e a k c a n b e a s s i g n e d r e a d i l y . T h e

s a m e p e a k c a n b e c o l le c t ed f o r E d m a n s e q u e n c-

i n g . T h e r e s u l t s f r o m E d m a n s e q u e n c i n g d e -

t e r m i n e t h e N - t e r m i n u s o f t h e p e p t i d e , w h e r e a s ,

t h e o b t a i n e d m a s s d e t e r m i n e s t h e C - t e r m i n u s o f

t h e p e p t i d e . T h i s p r o c e d u r e c a n b e a p p l i e d f o r

a ll H P L C p e a k s t o c o n f ir m t h e p r i m a r y s e q u e n c e

o f p r o t e in s . T h e d i s a d v a n t a g e o f u s i n g E S I - M S

i n p e p t i d e m a p p i n g i s t h e t i m e n e c e s s a r y f o r L C

a na lys i s , wh ic h c ou ld r a nge f r om 0 . 5 t o 2 h . F ig .

6 s h o w s a n e x a m p l e o f a n L C - M S a n al y si s o f t h e

t r y p s i n d i g e s t o f c a r b o x y a m i d o m e t h y l a t e d h G H .

B y e x am i n i n g t h e p e a k s i n t h e T I C , o n e c a n

a s s i g n t h e m a s s e s t o m o s t o f t h e H P L C p e a k s .

H o w e v e r , g l y c o p e p t i d e s o f t e n s h o w p o o r r e -

s p o n s e i n E S I - M S .

3.2.2 . Post translational modifications

A l a r g e n u m b e r o f p r o t e i n s u n d e r g o p o s t -

t r a n s l a t i o n a l m o d i f i c a t i o n a f t e r t h e i r b i o s y n t h e -

s i s . I n som e c a se s , pos t - t r a ns l a t i ona l m od i f i c a -

t i ons a r e c r i t i c a l f o r t he t r a nspo r t a nd b ioa c t iv i t y

o f p r o t e in s . P os t - t r a ns l a t i on a l m od i f i c a t ions a r e

o f t e n n o t d e t e c t e d i n E d m a n s e q u e n ci n g b e c a u s e

t h e m o d i f i e d r e s i d u e i s e i t h e r n o t d e t e c t e d o r i s

d e s t r o y e d u n d e r t h e h a r s h c h e m i c a l e n v i r o n -

m e n t . M a s s s p e c t r o m e t r y i s p r o b a b l y t h e b e s t

m e t h o d f o r t h e d e t e c t i o n o f p o s t -t r a n s la t i o n a l

m o d i f i c a t i o n s . K r i s h n a a n d W o l d [ 54 ] c o m p i l e d a

use f u l l i s t o f pos t - t r a ns l a t i ona l m od i f i c a t ions o f

p r o t e i n s . S o m e o f t h e c o m m o n p o s t - t ra n s l a t io n a l

m o d i f i c a t i o n s i n c l u d e a c y l a t i o n o f t h e N - t e r -

m i n u s , p h o s p h o r y l a t i o n o f se r i n e , t h r e o n i n e a n d

t y r o s i n e , o x i d a t i o n o f m e t h i o n i n e , g l y c o s y l a ti o n

o f s e r in e a n d a s p a r a g i n e , c l e a v a g e o f N - a n d / o r

C - t e r m i n a l r e s i d u e s b y p r o t e o l y t i c e n z y m e s a n d

f o r m a t i o n o f C - t er m i n a l a m i d e s . F o r s im p l e

m o d i f i c a t i o n s , s u c h a s a c e t y l a t i o n o r p h o s p h o r y l -

a t i on , t he a na lys i s i s s im p le a nd c a n be c a r r i e d

o u t u s i n g th e n o r m a l p e p t i d e m a p p i n g p r o c e d u r e

[ 55 ] . C a pr io l i e t a l . [ 56 ] de ve lope d a p r oc e dur e

t o g e n e r a t e a s e q u e n c e - o r d e r e d m a p f r o m a

p r o t e a s e d i g e s t b y u s i n g t h e o v e r l a p i n f o r m a t i o n

p r o d u c e d f r o m a t i m e - c o u r s e p r o t e a s e d i g e s t o f a

p r o t e i n . T h i s t e c h n i q u e i s b a s e d o n t h e f a c t t h a t

t h e r a t e o f c l e a v a g e c a n d i f f e r w i d e l y f r o m s i t e t o

s i t e a nd tha t a l l s i t e s a r e no t e qua l ly a c c e s s ib l e .

C l o s t r i p a i n w a s u s e d a s a l i m i t e d p r o t e o l y t i c

e n z y m e b e c a u s e i t h y d r o l y z e s o n l y a t t h e C -

t e r m in a l s ide o f a r g iny l re s idue s . S inc e no t a ll

a r g in ine s a r e a c c e s s ib l e t o t he e nz ym e , a t im e -

c o u r s e d i g e s t i o n w o u l d p r o d u c e p e p t i d e f r a g -

m e n t s w i t h l a r g e o v e r l a p p i n g r e g i o n s . O n e o t h e r

a p p r o a c h i s t o p e r f o r m t h e d i g e s t i o n i n o x y g e n -

1 8 - e n r i c h e d w a t e r ; o n l y t h e C - t e r m i n a l f r a g m e n t

wi l l be un la be l e d [ 57 ] .

P e r h a p s t h e m o s t c o m m o n m o d i f i c a t i o n o f

p r o t e i n s i s g l y c o s y l a t i o n . A n a l y s i s o f g l y c o p r o -

t e i n s b y m a s s s p e c t r o m e t r y i s c o m p l i c a t e d b y t h e

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32 D . N . N g u y e n e t a l. / J . C h r o m a t o g r . A 7 0 5 1 9 9 5 ) 2 1 - 4 5

° °

i 2 5 2 3

1

o

1 2 0 1 4 0 1 6 0 1 8 0 1

0 0 9 . 8 1 9 . 7 2 9 . 5 3 9 . 3

S c a n / ~ m e m i n )

1O 0

6 2 6 . 4

75

- 50

f f 26

o ~ = L L ~ t u . . . ~ J L i d . J . ~ ~ i J = . ~ = J [ _ _ t_ ~ L u t _ J . _

. . . . . . . . . . i . . . . . . . . .

4 0 0 6 0 0 8 0 0 1 00 0 1 2 0 0 1 4 0 0

m / z

Fig. 6. LC-M S o f a trypsin digest of carboxyamidom ethylated GH using ESI-MS. Peak 1 contains an ion at m z 626.4 which

matches the LED GSPR (T14) fragment of the protein. Ev ery peak in the TIC chrom atogramcan be analyzed n the same manner

to confirm the primary sequence of the protein.

h e t e r o g e n e i t y o f t h e c a r b o h y d r a t e m o i e t y . T h e

m o s t r e l i a b l e m e t h o d o f d e t e r m i n i n g t h e m o l e c u -

l a r m a s s o f a h e a v i l y g l y c o s y l a t e d p r o t e i n i s

M A L D I - T O F m a s s s p e c t r o m e t r y ; t h e c o m p l e x i t y

d e r i v e d f r o m t h e m u l t i p l e c h a r g e s t a t e s p r e s e n t

i n E S I - M S g e n e r a l l y r e n d e r s i t i m p r a c t i c a l f o r

t h i s p u r p o s e . H o w e v e r , w h e n t h e g l y c o p r o t e i n i s

d i g e s t e d i n t o s m a l l e r f r a g m e n t s , F A B , M A L D I

a n d E S I m a s s s p e c t r o m e t r y c a n b e s u c c e s s f u l l y

a p p l i e d t o t h e a n a l y s i s . A c l a s s i c a l a p p r o a c h t o

g l y c o p r o t e i n a n a l y s i s is t o d e g l y c o s y l a t e co m -

p l e t e l y u s i n g h y d r a z i n o l y s i s , i s o l a t e a n d p u r i f y

t h e p o l y s a c c h a r i d e s a n d t h e n p e r f o r m s t r u c t u r a l

c h a r a c t e r i z a t i o n . A m a j o r d r a w b a c k o f th i s t e c h -

n i q u e i s th a t s i te s o f g l y c o s y l a ti o n o n t h e p r o t e i n

c a n n o t b e d e t e r m i n e d b e c a u se o f th e d e s t r u c ti o n

o f t h e p r o t e i n . A p p l i c a t i o n o f m a s s s p e c tr a l

t e c h n i q u e s t o g l y c o p r o t e i n a n a l y s i s h a s p r o v i d e d

a m e a n s o f c h a r a c t e ri z i n g t h e se g l y c o p r o t e i n s

e f f i c i e n t l y . S e q u e n c e a n a l y s i s o f o l i g o s a c c h a r i d e s

i s o u t s i d e t h e s c o p e o f t h i s p a p e r , a l t h o u g h

S u t t o n e t a l. [ 58 ] h a v e u t i l iz e d M A L D I - M S t o

i d e n t i f y t h e s i te o f g l y c o s y la t i o n a n d t o o b t a i n

t h e s e q u e n c e o f t h e c a r b o h y d r a t e m o i e t y . T h e

i n i t i a l p r o b l e m w i t h a n u n k n o w n p r o t e i n i s t o

d e t e r m i n e w h e t h e r i t i s g l y c o s y l a t e d . O n e a p -

p r o a c h h a s b e e n t o a p p l y a h i g h l y s e n s i t iv e l e c ti n

s c r e e n w h e r e a r a n g e o f l ec t in s m u s t b e e m -

p l o y e d t o e n s u r e t h a t a ll o f t h e c o m m o n o l i g o -

s a c c h a r i d e s w i ll b e d e t e c t e d [5 9]. O n c e a p r o t e i n

i s c o n f i r m e d t o b e a g l y c o p r o t e i n , i t i s t h e n

f r a g m e n t e d c h e m i c a ll y o r e n z y m a t ic a ll y . H a w k e

e t a l . [ 6 0 ] h a v e r e p o r t e d t h a t , b y u s i n g a n a f f i n i t y

c o l u m n , m o s t o f th e g l y c o p e p t i d e s c o u l d b e

s e p a r a t e d . T h e r e m a i n i n g f r a c t io n s w e r e a n a -

l y z e d b y H P L C a n d s c r e e n e d a g a i n f o r g l y c o p e p -

t i d e s. T h e i s o l a te d g l y c o p e p t i d e s w e r e t h e n s u b -

j e c t e d t o m a s s s p e c t r a l a n a l y s i s . T h e d e t e r m i -

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D.N. Ng uyen e t a l . / J . Chromatogr . A 705 19 95 )21 -45 33

n a t i o n o f N - l i n k e d o l i g o s a c c h a r i d e s c a n b e p e r -

f o r m e d r e a d i l y w i t h a p u r e o l i g o p e p ti d e . D i -

g e s t i o n o f t h e o l i g o p e p t i d e w i t h p e p t i d e - N -

g l y c o s i d as e F ( P N G a s e F ) i n 50 % 1 8 0 - la b e le d

w a t e r a f f o r d s a p a i r o f s i gn a ls ( M H ÷ a n d M H ÷ +

2 ) d u e t o t h e p a r t i a l i n c o r p o r a t i o n o f o x y g e n - 1 8

i n t o t he / 3 -c a rboxy l g roup o f a spa r t i c a c i d [ 61 ] .

U n f o r t u n a t e l y , th e r e i s n o k n o w n e n z y m e w h i c h

c a n h y d r o l y z e O - l i n k e d o l i g o s ac c h a r id e s . C a r r e t

a l. [2 ] h a v e p r o p o s e d a n e l e g a n t p r o c e d u r e u s i n g

L C - E S I - M S t o i d e n t i f y s e l ec t iv e l y N - a n d O -

l i n k e d o l i g o s a c c h a r i d e s i n g l y c o p r o t e i n s . T h e

g l y c o p e p t i d e s a r e i d e n t i f i e d u s i n g a c a r b o h y d r a t e

m a r k e r : on . T h i s i s d o n e b y s te p p i n g th e o r i f i ce

p o t e n t i a l t o e n h a n c e t h e

low m/z

f r a g m e n t s , i n

p a r t i c u l a r

m/z

2 0 4 . T h i s e x p e r i m e n t a l l o w s o n e

t o c o n f i r m t h e p r e s e n c e o f a g l y c o p r o t e i n a n d t o

l o c a l i z e t h e g l y c o p e p t i d e f r a g m e n t s i n t h e e n -

z y m a t i c d i g e s t e d c h r o m a t o g r a m . B y p e r f o r m i n g

a p r e c u r s o r s c a n o f

m/z

204 , g l yc ope p t i de s

c a n b e s e l e c t i v el y d e t e c t e d i n t h e p r e s e n c e o f

o t h e r p e p t i d e s . I t s h o u l d b e n o t e d t h a t t h e

s p e c t r u m g e n e r a t e d u s i n g t h i s p r o c e d u r e c o r r e -

s p o n d s t o a l l p a r e n t i o n s t h a t h a v e d e c o m p o s e d

t o y i e l d

m/z

2 04 . O - L i n k e d g l y c o p e p t id e s ca n b e

d i f f e r e n t i a t e d f r o m N - l i n k e d g l y c o p e p t i d e s b y

a n a l y z i n g a s a m p l e t h a t h a s b e e n t r e a t e d w i t h

P N G a s e F . S i te s o f g ly c o s y l a ti o n c a n b e o b t a i n e d

b y M S - M S a n al y si s o f t h e g ly c o p ep t id e s . E d m a n

s e q u e n c i n g c a n a ls o b e u s e d t o d e t e r m i n e t h e s i te

o f g l y c o s y l a t i o n ( a b s e n c e o f s i g n a l w h e r e t h e

r e s i d u e i s g l y c o s y l a te d ) . A s c h e m a t i c d i a g r a m o f

a s t r a t e gy fo r g l yc opro t e i n a na l ys i s i s show n i n

Fig . 7 .

O n e s h o u ld r e m e m b e r th a t c a r b o h y d r a te s

o f f e r a r e l a t i v e l y s m a l l n u m b e r o f p r o t o n a t i o n

s i t e s . T h i s p o i n t i s v e r y i m p o r t a n t w h e n E S I - M S

i s use d fo r g l yc opro t e i n a na l ys i s . F o r e xa mpl e , i f

a l a rge o l i gosa c c ha r i de i s a t t a c he d t o a sma l l

p e p t i d e , t h e r e m i g h t n o t b e e n o u g h c h a r g e t o

b r i n g t h e i o n s i n to t h e m a s s r a n g e o f t h e q u a d -

r u p o l e a n a l y z e r . I n t h i s c a s e , M A L D I - M S i s a

g o o d a l te r n a ti v e m e t h o d .

3.2.3. Protein degradation products

S i n c e a l m o s t a l l d e g r a d a t i o n p r o d u c t s o f p r o -

t e i n s r e s u l t i n a c h a n g e i n m o l e c u l a r m a s s c o m -

p a r e d w i t h t h e o r i g i n a l p r o t e i n , m a s s s p e c -

l ternate

P a t h

I 1 R e d u c e / A l k y la t e

: i g e s t i o n

L C / E S I M S

I

Stepped Ori f ice Potentia l

t o ~ n h ~ n n am 1 7 9 n 4

U n k n o w n P r o te i n ]

~ L ~ t i n c r e e n

[ G I y o r o t e i n [

I O i g e s t i o I

i

I A f f in i ty C h r o m a t o g r a p h y I

] G l y c o p e p f i c le s ] I P e p t i d e s

t

G l v c o o e ~ t

d e s

L P L C

I P u r e G l y c o p e p t id e ]

L C / E S I M S / M S t . q l ~

r e c u r s o r S c a n o f m / z 2 04

J

A f te r P N G a s eF T r e a t m e n t

Fig. 7. Strategy for glycoprotein analysis (see Refs. [60] and

[62]).

t r o m e t r y c a n b e u s e d t o d e t e c t s u c h d e g r a d a t i o n

p r o d u c t s , i n p r i n c i p l e . H o w e v e r , m a s s d e t e r m i -

n a t i o n o f t h e i n t a c t p r o t e i n d o e s n o t y i e l d i n -

f o r m a t i o n a s t o t h e l o c a t i o n o f t h e c h e m i c a l

m o d i f i c a t io n . F u r t h e r , f o r l a r g e p r o t e i n s ( M r

g r e a t e r t h a n a p p r o x i m a t e l y 2 0 0 0 0 ) , m a s s s p e c -

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

o l u t i o n t o d e t e c t m o d i f i c at i o n s o f o n l y 1 u ( e . g . ,

a s a r e s u l t o f d e a m i d a t i o n ) . H e n c e t h e m o s t

c o m m o n u s e o f m a s s s p e c t r o m e t r y w i th r e g a r d t o

c h a r a c t e r i z a t i o n o f p r o t e i n d e g r a d a t i o n p r o d u c t s

i s t h e c o m b i n e d u s e o f p e p t i d e m a p p i n g a n d

m a s s s p e c t r o m e t r y . E x a m p l e s o f t h i s a p p r o a c h

i n c l u d e t h e c h a r a c t e r i z a t i o n o f d e a m i d a t e d a n d

m e t h i o n i n e s u l f o x i d e f o r m s o f h u m a n g r o w t h

h o r m o n e [6 3] , h y d r o x y l a m i n e c l e a v a g e p r o d u c t s

i n i n su l i n - l i k e g row t h fa c t o r I [ 64 ] a nd de a mi -

d a t e d f o r m s o f t u m o r n e c r o s i s f a c t o r [ 6 5 ] . I n

e a c h o f th e s e e x a m p l e s t h e a p p r o a c h u s e d w a s t o

g e n e r a t e p e p t i d e f r a g m e n t s ( u s u a l l y b y t r y p s i n

d i g e s ti o n ) , i s o la t e t h o s e p e p t i d e f r a g m e n t s w h i c h

d i f f e r e d f r o m t h e f r a g m e n t s a r i s i n g f r o m t h e

n o n - d e g r a d e d p r o t e i n a n d c h a r a c te r i z e t h e m o d i -

f i ed p e p t i d e f r a g m e n t s b y F A B o r E S I ( u s u a l l y

e m p l o y i n g M S - M S t o c o n fi r m t h e s e q u e n c e

m o d i f i c a t i o n w h i c h h a d o c c u r r e d ) . I n m o s t c a s e s,

N - t e r m i n a l a n a l y s i s b y E d m a n d e g r a d a t i o n i s

a l so e m p l o y e d to p r o v i d e c o r r o b o r a t i n g e v i -

d e n c e .

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34 D . N . N g u y e n e t a l. / J. C h r o m a t o g r. A 7 0 5 1 9 9 5 ) 2 1 - 4 5

3 2 4 Pro te in metabo l i t es

W r o b l e w s k i a n d c o - w o r k e r s h a v e u s e d E S I - M S

t o s t u d y t h e i n v i t r o m e t a b o l i s m o f h u m a n

g r o w t h h o r m o n e ( h G H ) [6 6] a n d t h e i n v i v o

m e t a b o l i s m o f d e s ( 6 4 , 6 5 ) - h u m a n p r o i n su l i n [ 67 ].

I n t h e s e e x a m p l e s t h e p r o t e i n i s p r o t e o l y z e d , n o t

b y t h e a d d i t i o n o f e x o g e n o u s p r o t e a s e s a s i n a

t y p i c a l p e p t i d e m a p p i n g e x p e r i m e n t , b u t b y t h e

a c t io n o f e n d o g e n o u s p r o t e a se s . A n a b u n d a n c e

o f i n f o r m a t i o n c o n c e r n i n g t h e a c ti v i ti e s i n v o l v e d ,

t h e m e t a b o l i c p r o d u c t s a n d p o t e n t i a l m e t a b o l i c

p a t h w a y s c a n b e o b t a i n e d f r o m s u c h e x p e r i -

m e n t s .

H u m a n g r o w t h h o r m o n e w a s i n c u b a t e d w i t h

p r e p a r a t i o n s o f ra t t h y r o i d g l a n d ; t h e p r o d u c t 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 H P L C a n d c h a r -

a c t e r i z e d b y a c o m b i n a t i o n o f E S I - M S a n d N -

t e r m i n a l E d m a n s e q u e n c i n g . T h e p r e d o m i n a n t

a c t i v i t y t h a t a c t e d o n h G H w a s f o u n d t o b e a

c h y m o t r y p s i n - l i k e s e r i n e p r o t e a s e , b i o c h e m i c a l l y

s im i l a r t o r a t m a s t c e l l p r o t e a se - I , w i th c l e a va ge s

o c c u rr i n g ex c lu s iv e ly a t T y r - P h e - L e u - X a a

b o n d s . T h e p r e s e n c e o f a c a r b o x y p e p t i d a s e a c -

t i v i t y w a s i n d i c a t e d b y t h e d e t e c t i o n o f m e t a b o -

l i t e s t ha t we r e t r unc a t e d by a s ing l e a m ino a c id

a t t h e C - t e r m i n u s . T h e s e q u e n c e o f e v e n t s le a d -

i n g t o t h e d e g r a d a t i o n o f h G H i n th i s sy s t e m w a s

f o u n d t o b e i n i t i a t e d b y a c l e a v a g e b e t w e e n

T y r 1 4 3 - S e r 1 4 4 t o p r o d u c e a t w o - c h a i n f o r m o f

t h e p r o t e i n . T h i s w a s f o l lo w e d b y t h e c l e a v a g e o f

t h e i n i t i a l t w o - c h a i n f o r m a t T y r 4 2 - S e r 4 3 ,

l i b e r a t i n g t h e N - t e r m i n a l p e p t i d e P h e l - P h e 4 2 .

S u b s e q u e n t e v e n t s o v e r a 4 . 5 h i n c u b a t i o n l e d t o

t h e d e g r a d a t i o n o f h G H t o a s e t o f m o r e t h a n 2 0

pe p t ide s w i th m a sse s a t o r be low 2300 [ 66 ] .

T h e m e t a b o l i s m o f d e s ( 6 4 , 6 5 ) - h u m a n p r o -

i n s u l i n w a s e x a m i n e d i n r a t s a f t e r s u b c u t a n e o u s

a dm in i s t r a t i on . C i r c u l a t i ng in su l in - l i k e im m uno-

r e a c t iv i t y i n t he p l a sm a 25 m in a f t e r i n j e c t ion

w a s e v a l u a t e d b y a n i o n - e x c h a n g e a n d r e v e r s e d -

p h a s e H P L C . B o t h t e c h n i q u e s i n d i c a t e d t h e

p r e s e n c e o f a m e t a b o l i t e c o m p r i s i n g 5 - 1 0 % o f

t h e c i r c u l a t i n g i m m u n o r e a c t i v i t y a n d h a v i n g t h e

r e t e n t i on c ha r a c t e r i s t i c s o f hum a n in su l in . Th e

r e m a i n d e r h a d r e t e n t i o n c h a r a c t e r i s t i c s o f

d e s ( 6 4 , 6 5 ) - h u m a n p r o i n su l i n . T h e p e a k s o f i m -

m u n o r e a c t i v e m a t e r i a l w e r e i s o l a t e d a n d t h e i r

s t r u c t u r e s d e t e r m i n e d u s i n g r e v e r s e d - p h a s e

H P L C a n d E S I - M S . T h e m a j o r c i r c u l a t i n g c o m -

p o n e n t c o - e l u t e d w i t h d e s ( 6 4 , 6 5 ) - h u m a n p r o -

i n s u l i n a n d h a d a n i d e n t i c a l m a s s s p e c t r u m . T w o

c i r c u l a t i n g m e t a b o l i t e s w e r e i d e n t i f i e d . T h e s e

m e t a b o l i t e s c o - e l u t e d b y r e v e r s e d - p h a s e H P L C

w i t h h u m a n i n s u l i n a n d d i a r g i n y l ( B 3 1 , 3 2 ) - h u m a n

insu l in a nd ha d m a ss spe c t r a i de n t i c a l w i th t he

s t a n d a r d c o m p o u n d s . T h e d a t a i n d i c a t e t h a t

p r o t e o l y t i c p r o c e s s i n g o f d e s ( 6 4 , 6 5 ) - h u m a n

pr o insu l in i nvo lve s a n in i t i a l t r yp t i c c l e a va ge a t

t h e c a r b o x y s i d e o f A r g B 3 2 , w i t h t h e f o r m a t i o n

o f h u m a n i n s u l i n b y t h e s u b s e q u e n t a c t i o n o f a

c a r b o x y p e p t i d a s e t o r e m o v e t h e A r g B 3 1 -

A r g B 3 2 d i p e p t i d e f r o m d i a r g i n y l ( B 3 1 , 3 2 ) - h u m a n

insu l in . The r e su l t s sugge s t t ha t som e o f t he

p h a r m a c o l o g i c a l a c t i v i t y o f d e s ( 6 4 , 6 5 ) - h u m a n

p r o i n s u l i n m a y b e m e d i a t e d i n p a r t b y ci r c ul a ti n g

insu l in - l i k e m e ta bo l i t e s [ 67 ] .

3 2 5 D i s u l fi d e b o n d d e t e r m i n a t i o n

M a n y p r o t e i n s , e s p e c i a l l y s e c r e t e d o r n o n -

c y t o p l a s m i c p r o t e i n s , c o n t a i n d i s u l f i d e b o n d s

b e t w e e n p a i r s o f p ro x i m a l c y s t ei n e s . T h e d i s u l -

f i de bonds p l a y a r o l e i n t he s t a b i l i z a t i on o f t he

t e r t i a r y s t r u c t u r e o f t h e s e p r o t e i n s . P r o t e i n s t h a t

a r e p r o d u c e d t h r o u g h r e c o m b i n a n t D N A t e c h -

n o l o g y a r e o f t e n r e f o l d e d f r o m s t r o n g d e n a t u r -

a n t s i n to b io log ic a l ly a c t ive f o r m s a nd i t i s

c r it i ca l t o d e t e r m i n e w h e t h e r th e c o r r e c t

c ys t e ine s a r e i nvo lve d in f o r m ing the d i su l f ide

b o n d s . L i k e w i s e , f o r n a t u r a l l y o c c u r r i n g p r o -

t e in s , i t i s im por t a n t t o c ha r a c t e r i z e t he d i su l f ide

p a i r i n g p a t t e r n . S o m e p r o t e i n s c o n ta i n f r e e

c ys t e ine r e s idue s , no t i nvo lve d in d i su l f ide

b o n d s , a n d i t i s i m p o r t a n t t o i d e n t i f y t h e s e

r e s i d u e s . F o r e x a m p l e , t h e p r o t e a s e c l o s t r i p a i n

c on ta in s a c ys t e ine a t t he a c t ive s i t e a nd th i s f r e e

th io l g r oup i s e s se n t i a l f o r t he a c t iv i t y [ 68 ] .

A l t h o u g h a n a l y t i c a l m e t h o d s h a v e b e e n d e -

v e l o p e d t o d e t e r m i n e t h e t h i o l c o n t e n t i n a

p r o t e i n [6 9 ], t h e d e t e r m i n a t i o n o f t h e l o c a ti o n s

o f d i su l f ide bonds i s m or e o f a c ha l l e nge . D i su l -

f i d e - c o n t a i n i n g p e p t i d e s c a n b e i d e n t i f i e d b y

t h e i r a m i n o a c i d c o m p o s i t i o n o r s e q u e n c e o n l y i f

t h e y a r e p u r i f i e d t o h o m o g e n e i t y . F o r a p r o t e i n

c o n t a i n i n g m a n y d i s u l f i d e b o n d s , t h i s a p p r o a c h

c a n n o t b e a p p l i e d . H o w e v e r , t o t a l c y s t e i n e c o n -

t e n t c a n b e d e t e r m i n e d b y c o m p l e t e r e d u ct i o n o f

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D . N . N g u y e n e t a l . / J. C h r o m a t o g r . A 7 0 5 1 9 9 5 ) 2 1 - 4 5 35

t h e d i s u l f i d e b o n d s w i t h a s u i t a b l e r e a g e n t ( D q ' F

o r / 3 - m e r c a p t o e t h a n o l a r e c o m m o n l y u s e d ) f o l -

l o w e d b y a l k y l a t i o n w i t h i o d o a c e t i c a c i d o r

i o d o a c e t a m i d e . T h e t o t al m a s s i n c re m e n t o f th e

i n t a c t p r o t e i n a f t e r t h i s p r o c e d u r e d i v i d e d b y t h e

m a ss o f t he a lk y la t i ng g r oup ( e . g . , 58 f o r a c e t -

a m i d e ) y i e l d s t h e n u m b e r o f c y s t e i n e s i n t h e

p r o t e i n . I f t h i s p r o c e d u r e i s r e p e a t e d , b u t w i t h -

o u t p r i o r r e d u c t i o n , o n l y t h e f r e e c y s t e i n e s , n o t

invo lve d in d i su l f ide bonds , a r e a lk y la t e d . I t

s h o u l d b e c a u t i o n e d t h a t s o m e p r o t e i n s m a y

h a v e f r e e c y s t e i n e s b u r i e d i n s i d e t h e t e r t i a r y

s t r u c t u r e , p r e v e n t i n g t h e m f r o m b e i n g a l k y l a t e d .

T h e r e f o r e , i t m i g h t b e n e c e s s a r y t o p e r f o r m t h e

a i k y l a t i o n i n s t r o n g d e n a t u r i n g c o n d i t i o n s .

A g a i n , t h e m a s s i n c r e m e n t d i v i d e d b y t h e m a s s

o f t h e a l k y l a t i n g g r o u p y i e l d s t h e n u m b e r o f f r e e

c y s t e i n e s i n t h e p r o t e i n . T h e d i f f e r e n c e b e t w e e n

t h e t o t a l n u m b e r o f c y st e in e s an d t h e n u m b e r o f

f r e e c y s t e i n e s d i v i d e d b y 2 ( t w o c y s t e i n e s p e r

d i s u l f i d e b o n d ) g i v e s t h e n u m b e r o f d i s u l f i d e

b o n d s . O n e o f t h e g r e a t e s t p r o b l e m s i n l oc a li z i ng

the d i su l f ide bonds i s d i su l f ide e xc ha nge . App l i -

c a t i o n o f m a s s s p e c t r o m e t r y t o s o l v e t h i s p r o b -

l e m o r i g i n a t e s f r o m t h e o b s e r v a t i o n b y Y a z d a n -

pa r a s t e t a l . [ 70 ] t ha t d i su l f ide bonds a r e r e duc e d

i n s i t u d u r i n g p e p t i d e a n a l y s i s b y F A B - M S .

T h e d e t e r m i n a t i o n o f th e c y s t e i n e s i n v o l v e d in

a d i s u l f i d e p a i r i s a c c o m p l i s h e d b y a p e p t i d e

m a p p i n g a p p r o a c h . T h e k e y to a p p l y i n g t h is

a p p r o a c h s u c c e s s fu l l y i s to i s o l a t e p e p t i d e s t h a t

c on ta in a s ing l e d i su l f ide bond . F o r l a r ge p r o -

t e i n s c l e a v a g e w i t h c y a n o g e n b r o m i d e c a n b e

u s e d t o g e n e r a t e l a r g e p e p t i d e f r a g m e n t s t h a t a r e

m o r e s u s c e p t i b l e t o p r o t e o l y t i c d i g e s t i o n . T h e

p r o t e i n o r l a r g e p r o t e i n f r a g m e n t s a r e t h e n

d i g e s t e d w i t h a s u i t a b l e p r o t e a s e . A l t h o u g h

t r y p s i n c a n b e u s e d , c a r e m u s t b e t a k e n t h a t

d i su l f ide i n t e r c ha ng e no t oc c u r . I f d i su l f ide i n t e r -

c h a n g e i s a p r o b l e m , a n a c i d p r o t e a s e s u c h a s

p e p s i n s h o u l d b e u s e d . F o l l o w i n g p r o t e o l y t i c

c l e a v a g e , t h e p e p t i d e s a r e i s o l a t e d a n d s u b j e c t e d

t o b o t h E d m a n s e q u e n c i n g a n d m a s s s p e c t r a l

a n a l y s i s . T h e p e p t i d e s t h a t c o n t a i n m u l t i p l e

s e q u e n c e s a r e c a n d i d a t e s f o r p e p t i d e s t h a t c o n -

t a i n d i s u lf i d e b o n d s . I f m o r e t h a n t w o s e q u e n c e s

a r e f o u n d i n a n i s o l a t e d p e p t i d e p e a k , t h i s

i n d i c a t e s t h e e x i s t e n c e o f m o r e t h a n a s in g l e

d i s u l f i d e b o n d a n d r e q u i r e s s u b f r a g m e n t a t i o n

w i t h a s e c o n d p r o t e a s e . E v e n t h e e x i s t e n c e o f

o n l y t w o s e q u e n c e s d o e s n o t g u a r a n t e e a s i n g l e

d i s u l f i d e b o n d i f c l e a v a g e h a s n o t o c c u r r e d

b e t w e e n t h e t w o c y s t e i n e s o f o n e d i s u l f i d e b o n d .

O n c e a s e t o f p e p t i d e s , e a c h p e p t i d e c o n t a i n i n g a

s ing l e d i su l f ide bond , i s ob t a ine d , t he d i su l f ide

b o n d s c a n b e a s s i g n e d u n a m b i g u o u s l y . I f t h e

p r o t e in be ing a na lyz e d ha s a f r e e su l f hydr y l , i t

w i l l be ne c e s sa r y t o a lk y la t e i t be f o r e s t a r t i ng the

f r a g m e n t a t i o n p r o c e d u r e .

3 2 6 Ligan d binding

I n t h e f i e l d o f d r u g d i s c o v e r y , a c o m m o n

t h e m e i s t h e i d e n ti f i c at i o n o f l o w - m o l e c u l a r m a s s

c o m p o u n d s t h a t b i n d v e r y t i g h t l y t o a t a r g e t

p r o t e i n . A n e x a m p l e o f t h i s is th e c o v a l e n t

a t t a c h m e n t o f p r o t e a s e i n h i b i to r s t o p r o t e a s e s ,

o f t e n a t t h e a c t i v e s it e o f t h e p r o t e a s e . C o -

v a l e n t l y b o u n d l i g a n d s c a n b e s t u d i e d u s i n g t h e

n o r m a l p e p t i d e m a p p i n g p r o c e d u r e . S a i l a n d

K a i s e r [ 7 1 ] u s e d p e p t i d e m a p p i n g a n d E S I - M S t o

l o c al i ze t h e s i te o f b i n d i n g o f m e t h y l 3 - ( 2 - m e t h y l -

1 - o x o p r o p o x y ) [ 1 ] b e n z o t h i e n o [ 3 , 2 - b ] f u r a n - 2 - c a r -

b o x y l a t e , a p o t e n t a n d h i g h l y s e l e c t i v e i n h i b i t o r

o f t h r o m b i n , t o t h e a ct i v e s i te o f t h r o m b i n . T h e y

w e r e a b l e t o s h o w t h a t t h i s c o m p o u n d f o r m e d a

s t a b l e a c y l e n z y m e c o m p l e x a t t h e a c t i v e s i t e

S e r - 2 0 5 w h i c h , a l o n g w i t h H i s - 4 3 a n d A s p - 9 9 ,

m a k e u p t h e a c t i v e s i te t ri a d o f t h r o m b i n .

A s i m i l a r a p p r o a c h w a s u s e d w i t h t h e v i r a l

e n z y m e , r h i n o v i r u s 3 C p r o t e a s e , t o s h o w t h a t a n

i n h i b i t o r o f t h is e n z y m e b o u n d c o v a l e n t l y t o t h e

a c t i v e s i t e C y s - 1 4 6 ( Z i m m e r m a n a n d B e c k e r ,

p e r s o n a l c o m m u n i c a t i o n ) . I n a d d i t i o n a l t o

l o ca l iz i n g th e s i t e o f a tt a c h m e n t , i n f o r m a t i o n o n

t h e n a t u r e o f t h e c h e m i c a l r e a c t i o n i n v o l v e d c a n

b e o b t a i n e d f r o m t h e i n c r e m e n t i n m a s s t h a t t h e

p r o t e i n u n d e r g o e s .

3 2 7 E nz ym e active si tes

A r e l a t e d a p p l i c a t io n i s t h e c h a r a c t e r i z a t i o n o f

e n z y m e a c t i v e s i t e s . T h e g e n e r a l a p p r o a c h i s t o

u t i l i z e a spe c i f i c a f f in i t y l a be l wh ic h f o r m s a

c o v a l e n t b o n d w i t h o n e o f t h e a c t i v e s i t e r e s -

i d u e s . P e p t i d e m a p p i n g i s t h e n a p p l i e d t o l o c a l -

i z e t h e p e p t i d e c o n t a i n i n g t h e m o d i f i c a t i o n .

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36 D . N . N g u y e n e t a l. / J . C h r o m a t o g r . A 7 0 5 1 9 9 5 ) 2 1 - 4 5

Several examples of this approach can be found

in the recent literature.

The apparent active site of human leukocyte

glycoasparaginase, an enzyme involved in the

degradation of the N-glycosidic linkage between

asparagine and N-acetylglucosamine in various

glycopeptides, has been identified by labeling

with an inhibitor, 5-diazo-4-oxo-L-norvaline, an

asparagine analog [72]. The labeled protein was

digested with trypsin and analysis of the peptides

by mass spectrometry revealed that the inhibitor

was attached through an a-ketone ether linkage

to the hydroxyl group of the N-terminal

threonine.

Inactivation of histidine ammonia lyase from

Pseudomonas putida was accomplished by treat-

ment with L-cysteine at pH 10.5 in the presence

of oxygen [73]. Inactivation was accompanied by

the formation of a new species with a UV

absorbance maximum at 340 nm. Following

trypsin and staphylococcal V8 protease diges-

tion, a peptide was isolated that was found to

contain a modification of mass 184 by ESI-MS.

The modification was localized to Ser-143 of this

enzyme and the authors conclude that this repre-

sents the site for attachment of an electrophilic

cofactor required for histidase activity.

3 2 8 Peptide mass maps

An innovative approach to the identification of

protein sequences in a large protein sequence

database such as the Protein Information Re-

source (PIR) or the SWISSPROT database has

been created by Yates et al. [74]. The approach

involves the proteolytic digestion of an unknown

protein and then analysis of the peptides by a

peptide mapping/MS approach. They have dem-

onstrated that a set of observed masses which is

less than 50% of the total number of predicted

masses can be used to identify a protein se-

quence in the database. Mass maps generated by

ESI-MS, MALDI-MS and FAB-MS should all

work with this approach. When multiple matches

are found, tandem mass spectrometry (discussed

in the next section) can be used to establish

sequence similarity.

3 3 Sequencing

3 3 I Tandem mass spectrometry

Tandem mass spectrometry (MS-MS) was

originally developed for the investigation of ion

chemistry. However, its major application cur-

rently is in determining the structure of bio-

molecules. The tandem mass spectrometer could

be a multisector magnetic instrument (tandem

double focusing) or a triple quadrupole instru-

ment. In both cases, the first mass spectrome-

ter (or the first quadrupole) serves as a mass

separator. The selected ion (precursor ion) can

be induced for fragmentation by colliding with

an inert gas. This process is often referred to as

collision-induced dissociation (CID). The frag-

ment ions, commonly referred to as product

ions, are analyzed in the second mass spec-

trometer (or the third quadrupole). A detailed

description of tandem mass spectrometers has

been given elsewhere [75]. This section will only

describe the use of tandem mass spectrometry in

peptide sequencing.

A diagram of tandem mass spectrometry is

shown in Fig. 8. A peptide mixture consisting of

R, M and N components is ionized. Peptide M is

then selected by the first mass analyzer and

subsequently induced by colliding with a neutral

gas such as argon or helium to give fragment ions

A, B and C. These fragment ions can be scanned

by the third mass analyzer to give the mass

spectrum of the product ions. The use of a

collision gas is essential to produce a significant

number of intense product ions.

In peptides, the fragmentation generally oc-

curs at the peptide backbone. However, cleavage

at peripheral bonds can occur, particularly when

using magnetic sector mass analyzers. For pep-

tides with mass greater than 2500, fragmenta tion

seldom occurs because the vibrational energy

Pept ide Mass Mass

m i x t u re I o n i z a t io n S e p a r a t i o n

Fragmenta t ion Sep ara t io n e tec t ion

R R + [ ~ A +

M . ~ I ~ . M + - - - . I P . - M + - - I ~ 'M + - - - - ' I ~ ' B + - - I D B + - I I ~ f N ~ ' ~

N N + I ~ C +

F i g . 8 . M S - M S o f R , M a n d N p e p t i d e m i x t u r e .

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D.N . Nguyen e t a l . / J . Chromatogr . A 705 1995) 21 -4 5 3 7

induced by collision is distributed through a

larger number of bonds (except for post-source

decay MALDI- MS where fragmentation of pep-

tide with molecular mass up to 3000 Da can

occur). In order to understand peptide frag-

mentation, a nomenclature system was proposed

by Roepstorff and Fohlman [76]. This system

was soon modified to use lower case rather than

capital letters to avoid confusion, e.g. C n could

be mistaken for a cysteine residue at position n

[77]. Table 3 shows the common types of frag-

ment ions.

The mechanism for fragmentation is not fully

understood at the present time. The formation of

a n, b n and Yn is generally accepted to involve

initial protonation of the amide nitrogen atom.

d n Ions retain the positive charge at the N-

terminus and are formed as a result of the loss of

an alkene from the side-chain of the other

terminus of a n ions. d , Ions provide a means to

differentiate the isomeric amino acids leucine

and isoleucine. If leucine were the amino acid at

the terminus, an isopropyl radical would be

eliminated. On the other hand, isoleucine would

eliminate a methyl or an ethyl radical [78,79].

Johnson et al. [80] proposed mechanisms for the

formation of the fragment ions. It is important to

know that these mechanisms are chemically

plausible, but there are few experimental data to

support them except for the observed ions. In

the most common form of ESI-MS, in which

quadruple mass analyzers are used, b and y

fragment ions predominate.

Tandem mass spectrometry has been used to

study a large number of peptides and proteins.

Some of the notable works include the studies of

bradykinin [81], substance P [82], somatost atin

[83] and thioredoxin [84]. So far, most of the

proteins studied are well characterized and have

known primary sequences. For an unknown

protein, the tandem mass spectrum can be very

complicated because some proteins exhibit both

N- and C-terminal fragments. There are also

fragments that do not fit in any of the proposed

fragment ions. Despite these complications, mass

spectrometry offers protein chemists a means to

obtain sequencing information much faster than

any other analytical method. There are also

T a b l e 3

C o m m o n t y p es o f f r ag m e n t i o n s

a A / o 4 ¢ d d 4 /

/ /~l

H O

I

N . ~ L ~ O H

R4

I o n T y p e o f c l e a v ag e T e r m i n u s S t r u c tu r e

a n B a c k b o n e N

b n B a c k b o n e N

c n B a c k b o n e N

x B a c k b o n e C

y n B a c k b o n e C

z ,, B a c k b o n e C

d n S i d e - c h a i n N

v S i d e - c h a i n C

w S i d e - c h a i n C

+ N H = C H R

- N H C H R C O +

- [ N H C H R C O N H 2 ] H ÷

[ O C N H C H R C O 2 H ] H ÷

H ; N C H R C O -

[ C H R C O N H C H R C O 2 H ] H +

[ - N H C H = C H R ' ] H +

[ H N _ - C H C O N H C H R C O 2 H ] H +

[ R ' C H = C H C O N H C H R C O 2 H ] H +

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38 D.N. Nguyen et al. / J. Chromatogr. A 705 1995) 21 -45

computer programs which can be used to assist

in the analysis of spectral data of peptides. Some

of the commonly used programs include Mac-

ProMass [85], Mac Mass [86] and COMPOST

[ 8 7 ] .

3 .3 .2 . Pos t s ource decay MA L D I M S

In addition to tandem mass spectrometry,

post-source decay (PSD) is a new technique for

peptide sequencing. It has been observed that

large peptide and protein ions are unstable on

their way through a time-of-flight instrument.

The metastable decay of these ions has been

detected using two-stage reflectron MALDI-MS.

The term post-source decay was introduced by

Spengler and co-workers [88-91] to describe the

delay ed fragmentation of the desorbed analyte

ions. The fragmentation was due to the multiple

collisions of analyte ions with matrix ions during

the early plume expansion and ion acceleration

followed by collisions with residual or admitted

gas molecules in the field-free drift region of a

MALDI instrument. Unimolecular decomposi-

tion of the precursor ion gives rise to fragmenta-

tion patterns which can be used to determine the

sequence of the peptide. The fragment ions

travel with nearly the same velocities as their

precursors, but with lower kinetic energies.

Thus, by applying a retarding field at the reflec-

tor, one can separate the product ions from the

precursor ions. Because of the lower kinetic

energies of the product ions, they do not pene-

trate into the retarding field as deeply as their

precursors; therefore, they leave the reflector

earlier and arrive sooner at the detector. PSD-

MALDI offers many advantages over conven-

tional tandem mass spectrometry. The in-

strumental sensitivity for product ions is at least

two orders of magnitude better than tandem

mass spectrometry owing to the high ion trans-

mission and high ion yield (20-80% of precursor

ion). Also, MALDI-MS offers a much longer

dissociation time range than a conventional colli-

sion cell which allows the activation and decay of

peptides of M r up to 3000. The fragmentation

patterns of PSD-MALDI are very similar to that

of the low-energy CID [92]. Typically, a,, b, ,

C,, a n- 17 , b n- 17 , x,, Yn and z, ions are

observed in a mass spectrum. The loss of 17 u is

due to loss of ammonia from arginine residues.

There are drawbacks associating with PSD-

MALDI which should be mentioned. First, mass

calibration can be difficult because one would

have to perform tedious calibration with known

precursor ion and product ion masses. There-

fore, calibration is normally done using com-

puter software. This software takes into account

the instrument geometry, electrical field parame-

ters and the flight times of precursor and product

ions to calculate the ion masses. Second, for

large peptides (M r above 1500), it is often

necessary to increase the laser irradiance to have

sufficient desorption. However, increasing the

laser irradiance causes a decrease in resolution

which ultimately increases the uncertainty in

mass assignment of these peptides. Third,

because of the complexity of the fragmentation

patterns and the lack of algorithms for cleavage

interpretation, analysis of PSD mass spectra

requires much more time than it takes to acquire

the mass spectrum. Despite these difficulties,

PSD-MALDI has gained acceptance in peptide

sequencing. For example, Zambias et al. [93]

utilized PSD-MALDI for the analysis of a co-

valently bound peptoid (modified peptide) to a

polymeric bead. Hoyes et al. [94] utilized PSD-

MAL DI for the analysis of ACTH peptide (M r

2466). Spengler and Kaufmann [ 95] utilized

PSD-MALDI to localize the site of a post-trans-

lational modification of a peptide. The analysis

of a lipopeptide (M r 1863) located the fatty acid

on the N-terminus of the peptide. One interest-

ing, perhaps very important, application of PSD-

MALDI is the possibility of using this technique

for the characterization of unknown proteins. Yu

et al. [96] performed PSD-MALDI on an un-

known tryptic fragment. The resulting primary

sequence of this peptide was compared with

existing sequences in a database to suggest that

this protein resembles CHO MCP-1 protein.

3.3 .3 . Carboxypept idases

Carboxypeptidases, exoproteases which cleave

only the C-terminal residues in proteins, have

been used in protein sequencing with variable

success. Four carboxypeptidases, carboxypepti-

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d a s e P ( C P P ) , A ( C P A ) , B ( C P B ) o r Y ( C P Y ) ,

o r a c o m b i n a t i o n o f t h e s e h a v e b e e n m o s t

c o m m o n l y u s e d i n s e q u e n c i n g a p p l i c a t i o n s . I n

g e n e r a l , t h e r a t e s o f c l e a v a g e b y t h e s e e n z y m e s

a r e d e p e n d e n t o n t h e p o l a r i t y o f t h e C - t e r m i n a l

a m i n o a c i d s i d e - c h a i n , m a k i n g s e q u e n c i n g p r o -

g r e s s e x t r e m e l y v a r i a b l e . B e c a u s e o f t h i s v a r i -

a b i l i t y , c a r b o x y p e p t i d a s e s h a v e t r a d i t i o n a l l y

b e e n u s e d t o d e t e r m i n e o n l y t h e C - t e r m i n a l

r e s i d u e o f a p r o t e i n b y i d e n ti f y i n g th e a m i n o a c i d

r e l e a s e d b y i ts c h r o m a t o g r a p h i c r e t e n t i o n t i m e

[97].

A d i f f e r e n t a p p r o a c h i s t o m o n i t o r t h e t r u n -

c a t e d p r o t e i n i n s t e a d o f t h e r e l e a s e d a m i n o a c i d

a n d t h e t e c h n i q u e s o f E S I - M S , P D - M S a n d

M A L D I - M S n o w m a k e t h i s a p p r o a c h f e a s i b l e . A

d i g e s t i o n r e a c t i o n i s c a r r i e d o u t u s i n g c a r b o x y -

p e p t i d a s e ( s ) o f c h o i c e. A t s e l e c t e d i n t e r v a ls ,

a l i q u o t s a r e r e m o v e d a n d t h e r e a c t i o n i s

q u e n c h e d a n d s t o r e d f o r l a t e r a n a l y s i s . U s i n g

P D - M S , t h e d i g e s t i o n c a n b e c a r r i e d o u t d i r e c t l y

o n t h e n i t r o c e ll u l o s e s u p p o r t [ 9 8 ,9 9 ]. W i t h E S I -

M S , t h e d i g e s t i o n m i x t u r e c a n b e m o n i t o r e d b y

c o n t i n u o u s i n f u s io n . S m i t h a n d D u f f i n [1 00 ] u s e d

c a r b o x y p e p t i d a s e P t o s e q u e n c e i n t e r l e u k i n 3

( I L - 3 ) a n d s u p e r o x i d e d i s m u t a s e ( S O D ) . I L - 3

h a s a C - t e r m i n a l s e q u e n c e o f . . . T F L S L A I F .

T h e s e q u e n c in g w a s p e r f o r m e d i n 1 5 m M a m -

m o n i u m a c e t a t e b u f f e r ( p H 4 . 0 ) . U n d e r t h e s e

c o n d i t i o n s , s ix a m i n o a c i d r e s i d u e s w e r e d i g e s t e d

b y C P P . A d d i t i o n a l r e a c t i o n t i m e d i d n o t r e l e a s e

a n y f u r t h e r r e s i d u e s . S u p e r o x i d e d i s m u t a s e h a s a

C - t e rm i n a l s e q u e n c e o f . . . C G V I G I A K . D i g e s-

t i o n o f t h i s p r o t e i n y i e l d e d o n l y o n e r e s i d u e .

H o w e v e r , a f t e r r e d u c i n g a n d a l k y l a t i n g t h e p r o -

t e i n w i t h i o d o a c e t i c a c i d , t h r e e r e s i d u e s w e r e

r e l e a s e d b y t h e e n z y m e . T h e s e q u e n c i n g d i d n o t

p r o c e e d p a s t t h e g l y c i n e r e s i d u e e v e n w i t h e x -

t e nde d r e a c t ion t im e . S c h / i r e t a l . [ 101 ] r e po r t e d

t h e u s e o f a m i x t u r e o f C P A a n d C P B a n d a l s o

C P Y f o r t h e d i g e s t i o n o f s y n t h e t i c p a r a t h y r o i d

h o r m o n e u s i n g M A L D I - M S . A f t e r a b o u t 2 h o f

d i g e s t i o n , 2 1 a m i n o a c i d r e s i d u e s w e r e r e m o v e d

f r o m t h e p r o t e i n . I t w a s f o u n d t h a t C P Y d i g e s t s

va l ine r a p i d ly a nd ve r y s lowly a t h i s t i d ine r e s -

idue s , he nc e i t wa s no t poss ib l e t o l oc a l i z e a l l t he

t r u n c a t e d p r o t e i n s . A l s o , t ru n c a t e d p e p t i d e s o f

M r b e l o w 1 20 0 w e r e d i ff i cu l t t o d e t e c t b e c a u s e o f

i n t e r f e r e n c e f r o m t h e m a t r i x . R o s n a c k a n d S t r o h

[ 1 0 2 ] d e s c r i b e d t h e u s e o f a l o w - f l o w r e a c t o r t o

m o n i t o r t h e d i g e st i on o f g l u ca g o n a n d a p o m y o -

g l o b i n u s i n g E S I - M S . T h e p r o t e i n a n d e n z y m e

( C P P ) w e r e m i x e d a n d i n f u s e d i n t o a f u s e d - s i l i c a

r e a c t o r w i t h c o n t i n u o u s m o n i t o r i n g b y E S I - M S .

G l u c a g o n g a v e s e q u e n c e i o n s f o r t h e f i r s t 1 9

a m i n o a c i d s ; h o w e v e r , a p o m y o g l o b i n g a v e o n l y

5 0 % o f t h e f i rs t 3 0 a m i n o a c i d s . A t t e m p t s w e r e

m a d e t o s e q u e n c e c y t o c h r o m e c a n d c a r b o n i c

a n h y d r a s e . U n f o r t u n a t e l y , n o s e q u e n c e i n f o r m a -

t i o n w a s o b t a i n e d f r o m t h e s e d i g e s t i o n s .

T h e a p p l i c a t i o n o f c a r b o x y p e p t i d a s e s t o p r o -

t e i n s e q u e n c i n g h a s b e e n i n v e s t i g a t e d i n o u r

l a b o r a t o r y . W e d e c i d e d t o u s e E S I - M S i n c o m b i -

n a t i o n w i t h C P P a n d C P Y f o r p r o t ei n s e q u e n c -

ing . G lu ca go n ( H S Q G T F T S D Y S K Y L D S R R A Q -

D F V Q W L M N T ) w a s u s ed a s a m o de l co m p o u n d

w i t h a m i x t u r e o f C P P a n d C P Y a s r e a g e n t s . T h e

d e t e c t i o n o f t h e t r u n c a t e d f o r m s o f g l u c a g o n w a s

c a r ri e d o u t b y E S I - M S . T h e c o m b i n a t i o n o f C P P

a n d C P Y w a s f o u n d t o b e n e c e s s a r y f o r s u c c e s s -

f u l d i g e s t i o n . F o r e x a m p l e , d i g e s t i o n w i t h C P Y

a l o n e w a s r e t a r d e d a t t h e t r y p t o p h a n r e s i d u e a n d

v i r tua l ly s toppe d a t t he a spa r t i c a c id r e s idue .

D i g e s t i o n w i t h C P P a l o n e p r o c e e d e d p a s t t h e s e

r e s i d u e s b u t w a s r e t a r d e d a t t h e l e u c i n e r e s i d u e .

A c o m b i n a t i o n o f C P P a n d C P Y r e s u l te d i n

d i g e s t i o n o f t h e e n t i r e g l u c a g o n m o l e c u l e i n

a bou t 70 m in . ( F ig . 9 ) . P e a k s a s soc i a t e d w i th a l l

o f t h e p r e d i c t e d g l u c a g o n f r a g m e n t s c o u l d b e

i d e n t i f i e d i n t h e m a s s s p e c t r u m t a k e n a t 7 0 m i n

a l t h o u g h t h e r e w a s a h i g h b a c k g r o u n d . O b v i o u s -

l y , t h e s e q u e n c e o f g l u c a g o n i s k n o w n a n d t h i s

g r e a t ly f a c i l i t a t e s t he a na lys i s o f t he m a ss spe c -

t r u m . A n u n k n o w n p r o t e i n w o u l d p re s e n t a

m u c h g r e a t e r c h a l l e n g e a n d a t p r e s e n t t h e t e c h -

n i q u e i s p r o b a b l y b e s t a p p l i e d t o t h e c o n f i r m a -

t i o n o f a k n o w n o r p r e d i c t e d s e q u e n c e . T h e

a b i l i t y t o o b t a i n s e q u e n c e i n f o r m a t i o n r e a d i l y

f r o m t h e C - t e r m i n u s o f a p r o t e i n i s , h o w e v e r , a n

i m p o r t a n t a d v a n c e i n p r o t e i n c h a r a c t e r i z a t i o n .

3 . 4 . N o n - c o v a l e n t i n t e ra c t i o ns

N o n - c o v a l e n t l y b o u n d l i g a n d s p o ~ e a p r o b l e m

i n t h e a n a l y s i s o f t h e s e c o m p l e x e s b y m o s t

i o n i z a t i o n t e c h n i q u e s . H e n i o n a n d c o - w o r k e r s

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40

D.N. Nguyen et al. / J. Chromatogr. A 705 1995) 21 -45

A

1 0 0

3 4 8 2 . 4

75

5 0 .

>=

m

2 5 -

0

2800

3 1 3 6 . 1

2 8 3 6 . 9

3 0 2 2 . 8

I I I

3 2 8 . 3 3 3 8 1 . 3

3 0 0 0 3 2 0 0 3 4 0 0

M o l e c u l a r W e i g h t

B .

/ 1 . = . .

1 0 0

7 5

v

.-=

ii)

E 5 0 ,

2 5 .

o

' + ~ e ~

. - . X L . X .

O

4 0 0

6 0 0 8 0 0 1 0 0 0

1 2 0 0

m , ' z

Fig. 9. Sequencing of glucagon using a mixture of CPP and CPY. (A) Reconstructed mass spectrum of digestion using CPP and

CPY after 15 min. Data were acquired in the chromatographic mode. (B) The actual ion m/z of digestion after 70 min; n refers to

the glucagon and n-18 refers to glucagon minus 18 amino acid residues from the C-terminus. The charges of the ions are shown in

parentheses. The charge is not specified when the ion is in a + 1 charge state. The + 1 charge states were assigned by examination

of the isotopic pattern of the peak. A total of 21 nmol of glucagon was consumed. See to Table 1 for peak assignments.

suggested that non-covalent molecular associa-

tion complexes might be detectable under the

soft ionization conditions offered by ESI and

that the reaction might be monitored in a real-

time mode [103]. The first successful application

of ESI-MS to the detection of non-covalent

receptor-ligand complexes was reported by the

same laboratory [104]. The macrolide FKS06, an

immunosuppressive agent, inhibits T-cell activa-

tion when in a complex with the cytoplasmic

receptor FKBP. FKBP was mixed with a slight

excess of FK506 at pH 7.5, and a new signal

appeared corresponding to the complex. Two

important controls were performed to ensure

that covalent adducts did not form. First, when

FK506 was combined with denatured FKBP, the

signal for the complex was not observed. Sec-

ond, when FK506 was replaced with a more

potent reagent (higher affinity toward the re-

ceptor), the peak corresponding to the new

complex was much stronger in intensity.

ESI-MS has also been used to study the

hydrolysis of a hexasaccharide of N-acetylglu-

cosamine (NAG6) by lysozyme. A mass spec-

trum immediately after mixing the enzyme and

substrate reveals a new protonated peak which

corresponds to the lysozyme-NAG6 complex.

During the time-course reaction, the spectrum

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D.N. Nguyen et al. / J. Chromatogr. A 705 1995) 21-45 41

shows the disappearance of lysozyme-NAG6

and appearance of lysozyme-NAG4 and

lysozyme-NAG3 [105].

Kata and Chait [106] demonstrated the utility

of ESI-MS in analyzing protein-ligand complex-

es by studying the heme-globin interaction. The

oxygen-carrying protein myoglobin contains a

non-covalently bound heme group in the hydro-

phobic pocket of the native globin chain. At low

pH values, unfolding of the protein results in

complete protonation of buried histidine res-

idues, resulting in a higher charge-state and a

loss of the heme group. A mass spectrum ob-

tained from an aqueous myoglobin solution at

pH 3.35 (completely denatured protein) shows a

single distribution of peaks and the total absence

of ions correspond to the heme-globin complex.

However, at pH 3.9; where both native and

denatured forms of myoglobin coexist, the mass

spectrum exhibits two distinct distributions of

peaks. One set of peaks corresponds to the

denatured myoglobin (higher charge state) and

the other set corresponds to the intact heme-

globin complex. These results suggest that na-

tive, non-covalent complexes of proteins and

cofactors in solution can be preserved in the gas

phase and observed by mass spectrometry.

Many proteins self-associate to form non-co-

valent dimers or higher oligomers. For example,

human growth hormone (hGH), under certain

conditions, will form a tightly complexed, non-

covalent dimer [107]. This dimer is stable under

aqueous conditions and can be resolved from the

mono mer by high-resolution size-exclusion chro-

matography. Re-chromatography of the isolated

dimer on the same system reveals that this

fraction is still dimeric with the retention time

expected for the dimer and has not reverted to

monomer. However, addition of 30% acetoni-

trile to the sample before re-chromatography

caused the conversion of the dimer into mono-

mer. Likewise, treatment with SDS sample buf-

fer followed by analysis by SDS-PAGE shows

that the dimer has been converted into mono-

mer. This hGH dimer has been studied by ESI-

MS (Fig. 10). Under the conditions normally

used to obtained protein spectra, 0.1-1.0%

acetic acid-50% acetonitrile, only the spectrum

®

r ,r

1581 .5

100

1476.1

75

1383 .9

1302 .6 ]

2 5

1 2 3 0 . 3 1 I

1 , s . 8 / ~ /

I L L L

~ 2 5 o 1 ~ o

1703.0

1844 .9

1770 .9

1~o

~ z

2012.4

2213 .4

1 9 2 4 . 9

2 o ' o 0 , . 2 . ' s o

Fig. 10. Spectrumof a non-covalentdimer of human growth

hormone. The spectrum was obtained on a Sciex API-III

instrument by infusingat 2.5/zl/min a sample of hGH dimer

at a concentration of 1 mg/ml in 0.1% acetic acid. The

m/z

values corresponding exclusively to the dimer are marked

with asterisks.

of monomeric hGH was observed. This is not

surprising, since 30% acetonitrile had been

shown to dissociate the dimer. However, by

omitting the acetonitrile and infusing a solution

at a concentration of 1 mg/ml in 0.1% acetic

acid, evidence for the dimer was obtained. The

spectrum appears to be an overlay of the spec-

trum of the dimer and the spectrum of the

monomer. To rule out the possibility that the

observed dimer spectrum was the result of dimer

formation in the mass spectrometer, a solution of

monomeric hGH was studied under identical

conditions and no evidence of dimer formation

was observed.

3.5. Protein folding~higher order structural

characterization

3.5.1. Deuterium exchange

While mass spectrometry is most commonly

used to assess the primary structure of proteins

or peptides, there are various experimental ap-

proaches available which can yield information

concerning protein folding and higher order

(secondary, tertiary or quaternary) structure.

One such approach is the determination of

hydrogen-deuterium exchange rates under con-

trolled solution conditions. In one application

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42 D.N . Nguyen et al . / J. Chromatogr. A 705 1995) 21 -45

t h e e x c h a n g e r a t e s o f th e v a r i o u s a m i d e h y d r o -

g e n s i n c y t o c h r o m e c w e r e d e t e r m i n e d u s i n g

H P L C - F A B - M S [ 1 0 8 ] . C y t o c h r o m e c w a s i n c u -

b a t e d i n 2 H 2 0 a t v a r i o u s t e m p e r a t u r e s . A f t e r

i n c u b a t i o n f o r p r e s e t t i m e i n t e r v a l s , t h e p r o t e i n

wa s t r a ns f e r r e d in to a so lu t ion in wh ic h

d e u t e r i u m e x c h a n g e i s v e r y s l o w ( p H 2 - 3 , 2 3 ° C )

a n d d i g e s t e d w i t h p e p s i n . T h e n u m b e r o f

d e u t e r i u m a t o m s i n c o r p o r a t e d i n t o e a c h o f t h e

p r o t e o l y t i c p e p t i d e s w a s d e t e r m i n e d b y H P L C -

F A B - M S . T h i s a p p r o a c h c a n p r o v i d e i n f o r m a -

t i o n r e g a r d i n g t h e s e c o n d a r y o r t e r t ia r y s t r u c t u r e

o f t h e p r o t e i n , s i n ce a m i d e s w h i c h e x c h a n g e

r e a d i ly ha v e a g r e a t e r so lve n t a c c e s s ib i l i t y t ha n

d o a m i d e s w h i c h e x c h a n g e s l o w l y . I n c a s e s

w h e r e a b i o s y n t h e t i c f o r m o f a p r o t e i n i s b e i n g

s t r uc tu r a l l y c ha r a c t e r i z e d , t h i s a pp r oa c h c a n be

e spe c i a l l y u se f u l i f a na t ive ( na tu r a l sou r c e ) f o r m

o f t h e p r o t e i n i s a v a i l a b l e a s a c o m p a r a t o r .

3 . 5 . 2 . P r o t e o l y s i s

A n o t h e r u s e f u l s t r a t e g y f o r p r o t e i n s t r u c t u r e

e va lua t ion i s l im i t e d p r o t e o ly t i c d ige s t ion . Th i s

a p p r o a c h i n v o l v e s d i g e s t io n o f th e p r o t e i n w i t h a

p r o t e o l y t i c e n z y m e , u n d e r c o n d i t i o n s s u c h t h a t

the r a t e o f p r o t e o lys i s i s su f f i c i e n tly s low to a l l ow

d e t e r m i n a t i o n o f t h e k i n e ti c s o f f o r m a t i o n o f th e

v a r i o u s p e p t i d e f r a g m e n t s . T h i s a p p r o a c h h a s

b e e n u s e d t o c o m p a r e t h e s t r u c t u r e s o f w i l d - t y p e

a n d r e c o m b i n a n t y e a s t ( m u t a n t ) c a l m o d u l i n s ,

u s ing ES I - M S [ 109] . I n t h i s s tudy , t he c a l -

m o d u l i n s w e r e d i g e s t e d w i t h t r y p s i n u s i n g v a r y -

i n g p r o t e i n / e n z y m e r a t i o s . A f t e r d i g e s t i o n f o r

se l e c t e d t im e in t e r va l s , t he r e a c t ion wa s t e r m i -

n a t e d b y t h e a d d i t i o n o f s o y b e a n t r y p s i n i n h i b -

i t o r . T h e p e p t i d e s o l u t i o n w a s d e s a lt e d a n d t h e

c h a r a c t e r i s t i c f r a g m e n t s w e r e d e t e r m i n e d b y

E S I - M S ( w i t h o u t c h r o m a t o g r a p h i c s e p a r a t i o n ) .

T h e u s e o f E S I - M S d i r e ct ly , w i t h o u t p ri o r H P L C

se pa r a t ion , m a k e s t h i s a pa r t i c u l a r ly r a p id a s sa y

m e t h o d . H o w e v e r , t h e la c k o f a n H P L C s e p a -

r a t i o n m a k e s t h e t e c h n i q u e m o r e s u s c e p t i b l e t o

m a t r ix i n t e r f e r e nc e ( e . g . , s a l t s ) a nd the qua n t i t a -

t i v e c a p a b i l i t y o f s u c h a m e t h o d i s t h e r e f o r e

r e l a t i ve ly l im i t e d . N on e the l e s s , th i s a pp r oa c h

pr ov ide s s ign i f i c a n t i n f o r m a t ion r e ga r d ing the

s t r u c t u r e o f t h e r e c o m b i n a n t p r o t e i n .

3 . 5 . 3 . C h a r g e s t a te

T h e c h a r g e - s t a t e d i s t r i b u t i o n o b s e r v e d f o r a

p r o t e in i n ES I - M S c a n p r ov ide s ign i f i c a n t i n -

f o r m a t i o n r e g a r d i n g p r o t e i n f o l d i n g . F o r e x a m -

p l e , E S I - M S h a s b e e n u s e d t o s t u d y t h e m e c h a -

n i s m o f r e f o l d i n g o f a c i d - d e n a t u r e d m y o g l o b i n

[ 110] . I n t h i s s tudy , t he ES I - M S a na lys i s wa s

c o n d u c t e d a t n e u t r a l p H , s i n c e t h e h e m e g r o u p

w a s d e m o n s t r a t e d t o r e m a i n a t t a c h e d t o t h e

p r o t e i n u n d e r t h e s e c o n d i t i o n s . M y o g l o b i n w a s

in i t i a l l y de na tu r e d in 10% a c e t i c a c id a nd the n

r e f o l d e d b y a d j u s t i n g t o v a r i o u s p H l e v e l s ( 5 - 8 )

b y a d d i ti o n o f a m m o n i a s o l u t i o n . T h e m o l e c u l a r

m a ss a nd c ha r ge - s t a t e d i s t r i bu t ion o f t he pa r ti a l -

l y , o r f u l l y , r e f o l d e d p r o t e i n w a s t h e n d e t e r -

m i n e d b y E S I - M S a n a l y s i s . B a s e d o n t h e p H

d e p e n d e n c e o f t h e o b s e r v e d m o l e c u l a r m a s s

( a f fe c t e d b y t h e p r e s e n c e o r a b s e n c e o f t h e h e m e

g r o u p ) a n d c h a r g e s t a t e d i s t r i b u t i o n , t h e E S I - M S

d a t a i n d i c a t e d t h a t m y o g l o b i n r e f o l d s i n t w o

m a j o r s t e p s . T h e u n f o l d e d p o l y p e p t i d e c h a i n fi rs t

r e fo l d s t o f o r m a n a t i v e - l i k e s t r u c t u r e w i t h o u t

t h e h e m e g r o u p , a n d t h e b i n d i n g c a v i t y o f t hi s

s t r u c tu r e t h e n b i n d s t h e h e m e g r o u p b y n o n -

c o v a l e n t i n t e r a c t i o n . T h i s e x a m p l e c l e a r l y d e m -

o n s t r a t e s t h a t E S I - M S c a n p r o v i d e u s e f u l i n -

f o r m a t i o n r e g a r d i n g p r o t e i n f o l d i n g p a t h w a y s ,

a n d t h i s i n f o r m a t i o n c a n b e u s e d i n c o n j u n c t i o n

w i t h o t h e r m o r e t r a d i t i o n a l t e c h n i q u e s ( e . g . ,

c i r c u l a r d i c h r o i s m ) t o o b t a i n a m o r e c o m p l e t e

u n d e r st a n d in g o f t h es e c o m p l e x p h e n o m e n a .

3 . 5 . 4 . C r o s s - l i n k i n g r e a g e n t s

A d d i t i o n o f c ro s s - li n k i n g r e a g e n t t o p r o t e i n s ,

f o l l o w e d b y s u b s e q u e n t s t r u c t u r a l c h a r a c t e r i z a -

t i o n o f th e l i n k e d d o m a i n s , h a s b e e n a t ra d i t i o n a l

a p p r o a c h u s e d t o ' p r o b e p r o t e i n s e c o n d a r y a n d

t e r ti a r y s t r u c t u r e . M a s s s p e c t r o m e t r y c l e a rl y c a n

p r o v i d e a n a d d i t i o n a l a n a l y t i c a l t o o l t o b e u s e d

in t h i s a pp r oa c h . The in t e r f a c ing dom a ins i n

r e c o m b i n a n t h u m a n e r y t h r o p o i e t i n ( E P O ) h a v e

be e n c ha r a c t e r i z e d in t h i s m a nne r [ 111] . I n t h i s

c a s e , a m i n o g r o u p s w e r e s e l e c t i v e ly c r o ss - l i n k ed

by spe c i f i c c r os s - l i nk e r s suc h a s d i suc c in im idy l

s u b e r a t e o r d i t h i o b i s ( s u c c i n i m i d y l p r o p i o n a t e ) .

T h e l i n k e d r e g i o n s w e r e t h e n c h a r a c t e r i z e d b y

t r y p s i n d i g e s t i o n f o l l o w e d b y H P L C s e p a r a t i o n .

T h e i s o l a t e d t r y p t i c p e p t i d e s w e r e c h a r a c t e r i z e d

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D.N. Nguyen et al. / J. Chromatogr. A 705 1995) 21-45 43

b y s e q u e n c i n g ( E d m a n d e g r a d a t i o n ) a n d m a s s

s p e c t r om e t r y . T h e s e d a t a we r e u s e d t o d e t e r -

m i n e wh i c h l y s i n e gr ou p s i n E P O we r e ad j ac e n t

i n t h e t h r e e - d i m e n s i on a l s t r u c t u r e . T h e s t u d y

a l s o d e m on s t r a t e d t h a t n on - g l yc os y l a t e d an d

g l y c o sy l a t ed f o r m s o f E P O h a v e a h ig h d e g r e e o f

s i m i l ar i t y w i t h r e gar d t o t h e p r o t e i n c on f or m a-

t i on . A s i m i l ar ap p r oac h h as b e e n d e s c r i b e d f or

s t u d y i n g t h e s u b u n i t a s s oc i a t i on o f p r o t e i n s

[112] . In th i s case the prote ins are cross - l inked

wi t h g l u t ar a l d e h yd e an d t h e m o l e c u l ar m as s o f

t h e a d d u c t i s d e t e r m i n e d b y M A L D I - M S . T h e

v a l i d i t y o f t h i s ap p r oac h was c on f i r m e d b y

analyz ing a var iety of prote ins hav ing var ious

k n own s t a t e s o f a s s oc i a t i on .

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