Biomass toward fine chemical products: Oxidation of α-pinene over sieves nanostructured modified with vanadium

[

27]

identiﬁcation of histidine phosphorylations

549

[

27] Identification of Histidine Phosphorylations

in Proteins Using Mass Spectrometry and

Affinity‐Based Techniques

B y A NDR EW R . S . ROSS

Abstract

His ti di ne phosph or ylatio n p lays a k ey r ole in pr okaryotic s ignali ng and

a ccounts for a pproximat ely 6% o f t he p rot ein p hos pho rylati on events i n

e ukar yotic s. Phosphoh is tidines generally act as in term ed iates i n the trans fer

of phosphate g roup s f ro m d on or t o accept or molecul es . Exampl es i nclud e the

ba cter ia l phosphoeno lp yr uvat e:sugar ph os pho trans ferase s ys tem ( PT S) and

th e his tidine ki nases f oun d in t wo‐compo nent s ignal trans du ctio n path ways .

The l atter a re util ized by b acteria and plan ts to s ens e and adapt to changing

e nv ir onmental c ond itions . Des pit e t he impo rt ance o f hi stidi ne p ho sph or yl a-

ti on i n two‐c omponent s ignaling s ys tems , r elatively few pr otei ns h ave s o far

been identiﬁed a s cont ainin g ph os pho rylated h is tid ine r es id ues. Th is i s largel y

due to the instabilit y of p ho sph oh is tidin es , w hich, u nl ike the ph os pho es ters

fo rmed by s er ine, t hr eon in e, and t yr os ine, are lab il e and s us ceptib le to aci d

hydr ol ys is . N ev ertheless , it is po ss ible t o pr es erve and i dentify p ho sph or y-

la te d hi stidi ne r es id ues in t ar get pr ot eins us in g a ppr opr iate s am pl e p repara-

ti on, a fﬁnity pur iﬁcat ion, and mas s s pect rometri c techn iques. T his chapter

pr ov ide s a br ie f over view of s uch t echn iques, descri bes their u se in conﬁ rm ing

hi stidi ne phos phor ylat ion of a kno wn PTS pr ot ei n ( HPr ), and s uggests ho w

th is a pproa ch might be adapt ed f or lar ge‐s cale i dentiﬁcation o f h is ti din e‐

phosphor yla te d prot ein s in t wo‐compo nent s ys te ms .

Introduction

R ev er si bl e ph os pho rylation is on e of t he mos t com mon and mo st im por -

ta nt mec ha ni sms by w hich t he s tr uctu re and fu nc ti on o f a p rotei n can becom e

po s t‐tr ans la ti ona ll y mod iﬁed. I t is est imat ed t ha t up to 30% o f all pr otei ns

may be phosphor ylat ed at any given t ime (C ohe n, 2000) . Si te‐s peci ﬁc ph os -

phory la ti on of pr ot eins aff ects lo calization , t ur nover , and enzym atic activity,

a s wel l a s i nter ac ti on s w it h ot her p rot eins and DNA ( Patel and G el fand, 1996;

W ol sc hin e t a l., 2 005) . Ph os pho rylation occu rs on the s ide chains o f cer tain

a mi no a cid r es idu es . The r es ulting p ho sph oami no aci ds fal l in to thr ee cate-

g or ie s: O‐phos phates, w hich are f or med b y s er in e ( Ser) , thr eo ni ne ( Th r) , and

ty ros ine ( Ty r) a nd con tain p hos pho es ter linkages; N‐p ho sph ates for med by

M E T H O D S I N E N Z Y M O L O G Y , V O L . 4 2 3

2 0 0 7 , H e r M a j e s t y t h e Q u e e n i n r i g h t o f C a n a d a .

0 0 7 6 - 6 8 7 9 / 0 7 $ 3 5 . 0 0

D O I : 1 0 . 1 0 1 6 / S 0 0 7 6 - 6 8 7 9 (0 7 ) 2 3 0 2 7 - 7

#

5

50

genome‐wide analyses of two‐component systems

[27]

hi stidi ne ( Hi s) , l ys ine ( Lys) , and arginine ( Ar g) , which contain p hos pho am i-

da te bonds; a nd t he acyl‐p hos phate f or med b y aspartic aci d ( As p) . T he

a ddi ti on a nd r emoval o f pho sph at e grou ps are u suall y m edi ated by s peciﬁ c

class es of e nz ymes , s uch as hist idine kinases ( ph os pho rylati on of H is ) and

s erine/ thr eoni ne pho sph at as es ( deph os pho rylati on o f S er and T hr ). Pho sph o-

hi stidi ne s, whi ch are t he least s table of t he ph os pho am ino aci ds , m ay o r m ay

no t r equir e hi stidine p ho sph at as es , d ep end ing on th e pr otei n and, i n particu-

l ar , the r es idue s adjacen t t o p ho sph ohist id in e (Kl um pp and K riegl stei n,

2

00 2) .

I n te r ms of phy s io lo gi ca l f un ct io n , t he r e ar e tw o m ai n cl as s es o f p r o tei n

p ho s p ho r y l a ti on . Th e ﬁ r s t o f t he s e en c omp a ss e s p h o s ph o r yl at io n fo r th e

p ur p o s e of r e g ul a t i n g en z yma ti c ac ti vi t y, an d u s u a l ly i n vo l ves m od i ﬁ cat io n

o f Se r , T hr , a nd T yr r es i d u es . Th e r es u lt in g p h o s p h o es ter s ar e s ta bl e e n ti -

t ie s, a nd g e n e r a ll y s er v e t o r eg u l at e en z ym a ti c cat al ys i s wi th o u t d i r ec t

i nv ol v e me n t i n the ca ta ly t ic me ch a n is m. R ev er s i b l e, m ul t i s i te p h o s p h o r yla -

t io n of Se r , T hr , a n d Ty r me di at es n um er o u s si gn a l tr a ns d u cti o n p ath w ays

i n e uk a r y ot i c c e ll s ( C oh e n, 20 00 ) . Th e s ec on d c la s s en co m pa s s es p h o s p h o r -

y l a ti on fo r th e pu r p o s e o f p ho s p h at e gr o u p tr a ns fe r , an d i s gen er al l y

r e s tr i c te d to ph o s p h or y la t io n o f Hi s r es i du e s . Ph o s p h o h is t id i n es act as

h ig h ‐e ne r g y i nte r me d ia te s in t he t r an s f er o f p ho s p h ate fr o m p h o s p h o d on o r

t o ph os p h oa c c e p to r mo l ec u le s ( S to ck et al ., 19 89 ) , a r o l e fo r wh i ch th es e

l a bi l e mo di ﬁc a ti o n s ar e w ell s u it ed . E xa m p le s i n clu d e th e b act er ia l p h o s -

p h oe no l py r u v a te :s u ga r p ho s p h o tr a n s fe r a s e s ys tem ( Me ad o w et al . , 199 0 )

a nd th e hi s ti d i ne k in as e en zy mes f ou n d in t wo ‐co m po n en t s i gn al tr a ns d u c-

t ion pa t hw a y s ( Pa r ki ns o n an d K o fo id , 19 92 ) . T h e l at te r ar e u ti l ize d b y

b ac te r i a , pl a n ts , an d lo w er eu k ar y o te s t o s en s e an d ad ap t to ch an gi n g

e nv i r on me n t a l c on d it io n s ( K lu mp p an d K r ie gl s te in , 200 2 ) . I n b act er i a,

s uc h a da p ta t i on s ma y in cl u de ch an g es in mot il i ty, cel l m or p h o l og y, an d

g e ne e x pr e s s i on , as w ell as t he es t a bl is h me n t o f vi r u le n ce an d an ti b i ot ic

r e s i st a nc e .

Due to the impo rt ance o f pr ot ein p ho sphor ylatio n in r egul ating key

bi ologi ca l proc es ses, cons iderable eff or t has been pu t i nto develop in g pr oce-

dure s for identifyin g and map ping s it es of pho sph or ylatio n, bo th for in di vi du-

a l p r ot e i ns a n d o n a p r o t eo m e ‐w ide s cale ( B eausol ei l et al. , 2004; d e l a Fuente

v an Bentem e t a l. , 2006; N u¨ h se et al., 2003) . How ever , th e s ub stoi chi om etri c

natur e of thi s modiﬁcat io n cont inu es t o p resent m aj or chall en ges; i nd eed, the

ph os phory la te d f or m o f a p ar ticular pr otein m ay r epr es en t on ly a s mall

f raction of i ts total abun dance. Fu rt hermor e, m an y p rotei ns can u nd er go

ph os phory la ti on at diff er ent s it es (C oh en, 2000) , r es ultin g i n a n um ber o f

po te ntia l phosphor ylated isof or ms of w hich s ev er al m ay b e p resent at any

g iv en time. As a r es ult, t he f ull complement of ph os pho rylated p rotein s in

a cel l, tis sue, or organism ( th e p ho sph op rot eo me) can be extremely com pl ex.

[

27]

identiﬁcation of histidine phosphorylations

551

Fo r Hi s‐ph os p ho r y la t ed p r ot e in s , t he s it u at i o n i s fu r th e r co m pl i cate d b y

t h e fa c t th a t ph os p h o hi s ti d in es ar e s u s cep t ib l e to h y d r o ly s is u n d e r th e

a ci di c c ond i ti on s n or m al ly u s ed in p ho s p h o p r o tei n an d p h o s p h o pe p ti d e

a n a l y s is ( He s s e t al ., 19 88 ; M at t h ew s , 19 95 ) . I n co n tr a s t, th e p h o s ph o es te r s

f o r me d by Se r , Th r , an d Ty r r es i du e s r es i s t a c id tr e at m en t an d s o r em ai n

i nta c t du r i ng e x t r act io n , p ur i ﬁ c at i on , an d an a l yti cal p r o ced u r e s . Co n s e -

q u e ntl y , ph os ph o hi s t id i ne s ge n er a ll y go u nd e t ect ed i n co n ven ti o n al s tu d i es

o f pr o te i n ph os ph o r yla t io n ( K lu mp p an d K r ie g l s te in , 200 2 ) . Be fo r e d eci d -

i ng up on a s tr a te gy f or la r ge ‐s ca l e id e nt iﬁ ca t io n o f h i s ti di n e p h o s p h o r yl a-

t ion s , i t i s th e r e fo r e n ec es s ar y t o u nd e r t ak e a cr i ti cal r evi ew o f exi s ti n g

t e chn i qu e s fo r ta r g et i n g an d an a ly z in g p ho s p ho p r o te in s an d s i tes o f p r o tei n

p h os p ho r y l a ti on . S uc h t ech n i q ue s in cl u de s amp l e fr a c ti o n ati o n , af ﬁn i ty

p u r i ﬁc a ti on , g e l s ep a r at io n , an d ma s s s p ec tr o m et r y ( MS ) ‐b a s ed an al yt ic al

p r o ce d ur e s .

Sample Fractionation

As a ﬁr s t s te p in ad d r es s in g t he lo w r el at i v e ab u n d an ce o f p h o s p h o r y-

l a t e d pr o te i ns , i t is ad v is ab l e t o u s e s o me f or m o f fr a cti o n ati o n to r ed u ce

s a m pl e c omp l e x i t y d ur i n g s u bs e q u en t an a ly ti c a l s te ps ( G r uh l er et al . , 200 5 ) .

D iff e r e nt i a l c e ntr if u ga ti o n is a p op u l ar t ech n i q u e fo r fr a c ti o n ati o n o f

b io l og i c a l s a mp l es an d h a s b ee n ap p li ed s u cce s s fu l l y to p h o s ph o p r o teo m i c

s tu di e s ( N u¨ hs e e t al ., 20 03 ) . Pr o te i n s an d o r ga nel l es d i ffer i n s i ze, s h ap e, an d

d en s i ty , a nd c a n t he r ef o r e b e r es o l ve d f r om ce l l l ys at es o r ti s s u e h o mo ge -

n at e s by v a r y in g t he s p ee d o f ce n tr if u ga ti o n . T h e fo r ce s cr ea t ed at l o w

s p ee d s a r e s ma l l ( e. g., 60 0 ꢀg ) an d o nl y ve r y l ar ge o r d en s e p ar ti cl es wi l l

p r ec i pi ta t e , fo r m in g a p el le t f r om w hi ch p r ot ei n s can b e ext r acte d an d / o r

f u r the r pu r i ﬁe d . At h ig h s p ee d, mo s t p ar t icl es wi l l p r eci p i tate an d o n ly

s o lu bl e pr o te i ns w ill r em a in in s o lu t io n . Fig u r e 1 s h o ws a s te pw i s e fr a c ti o n -

a t ion pr o to co l for eu k ar y ot e s b as e d u po n d if fer e n ti al cen t r i fu gat io n . T h is

f ra c ti on a ti on te c h ni q u e ca u se s mi n ima l d is r u pt io n to th e s am p le an d i s ,

t h e r e for e , l i ke l y t o p r es e r ve la b ile mo d i ﬁ cat i on s, s u ch as p h o s p h o hi s t i d in es .

H o we v e r , i t s ho u ld b e b or n e in mi n d t ha t th e d en at ur i n g b u ffe r s an d

p h os p ha ta s e i nh i bi to r s n or m al ly u s ed t o s u pp r es s en zy m e act iv i ty d u r in g

p r o te i n e x tr a c ti on w ill n ot gu ar d ag ai n s t h y d r o ly s is o f p h o s p h o hi s ti d i n es .

Subfra ctiona tio n met ho ds ( e.g., f or r es olving m itochon dr ial and m icro-

s om al pr otei ns ) have als o been d evelo ped t o fur th er s im pl ify pr otei n and

pr ote ome a na ly sis (Hans on et al., 2001; N u¨ h s e et al. , 2003) . L inear and d is -

con ti nuous s uc rose dens it y gradien t f ractionation p rocedu res have tradi ti on-

a lly be en use d to pu rify plas ma membr anes and m itocho nd ri al com plexes

(H od g e s e t a l . , 1 97 2; V an Pu t te an d Pa tt er s on , 200 3 ) . H ow e ver , th es e

m eth od s a r e g e ne r all y t ime ‐co n s um in g , r es u lt i n l o w yi el d s , an d ar e p r o n e

5

52

genome‐wide analyses of two‐component systems

[27]

Tissue

Homogenize

Homogenate

Centrifuge @ 600×g for 20 min

Centrifuge supernatant @

3000×g for 20 min

Pellet 1

nuclei, whole cells)

(

Centrifuge supernatant @

0,000×g for 20 min

2

Pellet 2

mitochondria, some lysosomes

and peroxisomes)

(

Centrifuge supernatant @

Pellet 3

100,000×g for 60 min

(

lysosomes and peroxisomes,

some mitochondria)

Pellet 4

crude microsomes)

Final supernatant

(soluble proteins)

(

FIG . 1. S amp le fr ac tio n at io n b y d if fe r en tia l c en t r ifu g at io n (c o ur t es y of D r. U ma K . A ry a l,

N RC Pl an t Bio tec h n olo g y I n s ti tu t e) . P las ma me mb r ane p r o t ei ns in c ru de m icr os om a l f ra c t ion s

ma y b e fu r th er r es o lv ed b y a qu e o us t wo‐p h as e p ar t it io n in g .

t o c ont a min a ti on . Af ﬁ ni ty p ur iﬁ ca t io n o f p la s ma m emb r an e s fr o m cr u d e

m i cr os o m a l pe l l e ts h as b ee n ac h ie ved u s in g an aq u eo u s tw o ‐p h a s e s ys t em

co mp r i s i ng po l y et h yl e n e gl yc o l ( PE G) an d d e xt r an ( N u¨ h s e et al . , 200 3 ;

S chi n dl e r e t a l ., 20 06 ) . W h en mi xed , t he s e t w o p o ly mer s s ep ar a t e i n to

up pe r ( PEG) a nd lo wer ( dext ran) p hases. S ubs equ ent p ar ti ti on in g o f ani oni c

s pe c i e s i nto th e lo w er p ha s e r es u l t s in t he lat ter ’ s b e co m in g n eg at iv ely

ch a r g e d. T hi s , i n t ur n , ca u se s p la s m a me mbr a n es to m i gr at e i n to th e u p p e r

o r i nte r ph a s e r e g io n s , e f fe ct in g s ep a r at io n o f s ub ce ll u l ar m emb r an es o n th e

b as i s of c ha r g e r at h er t ha n d en s it y ( La r s s on et al . , 198 7; Per s s o n an d Jer g il ,

1

9 9 2 ) . As we l l as en h an c i n g d et ec t io n o f p ho s p h o r yl ated an d o th er l o w

a bu n da n c e pr o te i n s , f r act i o na t io n b y d if f er e nt ia l cen t r if u gat io n an d tw o ‐

p h a s e pa r t it io ni n g ar e ve r y u s ef u l f or s u bc ell u l ar l o cal i zat io n o f p r o tei n s ,

co mp l e me n ti ng i nf o r ma ti cs t oo l s s u ch as Ta r ge t P ( Em an u el s s o n et al. , 200 0 )

a nd Si g n a l P ( Ba n n ai et al ., 20 02 ) t ha t ar e al s o ava il ab l e fo r th i s p u r p o s e.

Tw o ‐ph a s e pa r t it io n in g ma y al s o as s i s t in t he d e te cti o n o f H is ‐co n tai n i ng

[

27]

identiﬁcation of histidine phosphorylations

553

p r ot e in s , wh i ch m ig ra t e p r ef e r en ti al ly in t o t he PEG p h as e (Wu en s ch el l

et al ., 1 9 9 0 ) .

Phosphoprotein Enrichment

R e g a r dl e s s of w he t h er o r n ot s am pl e f r acti o n ati o n p r o ced u r es ar e

e m pl oy e d , th e pu r iﬁ c at i on an d an a ly s is o f p ho s p h o pr o t ei n s can b e gr ea t l y

e n ha n c e d us i n g af ﬁ n i t y‐b as e d t ech n i q ue s . M o n o cl o na l an ti b o d ie s r ai s ed

a g a i ns t s pe c i ﬁ c p r ot e in mo d iﬁ ca t io ns ( e. g., p h o s ph o r yl ate d S e r , T h r, o r

Ty r r e s i du e s ) c a n b e u s ed t o is o la t e p r ot e i n s c ar r y i n g th es e m od i ﬁ cat io n s ,

e i th e r by i m mu n oa f ﬁ ni t y ch r o m at o gr a p hy o r i m m un o p r ec i p it at i o n ( L iu

et a l., 2 0 0 4 ) . Su c h an t ib o d ie s ca n al s o b e u s ed t o vi s u a l iz e m od i ﬁ ed p r o tei n s

s e pa r a te d on one ‐ o r t w o ‐d ime ns i o na l el ec t r o p ho r e s i s gel s ( s ee l ate r ).

A n ti bo dy pu r i ﬁc at io n o f p r ot e in s co n t ai ni ng p h o s ph o r yl ate d S er an d T h r

r e s i due s ha s me t w it h li mit e d s u cce s s ( Gr ø n bo r g et al ., 200 2 ) , d u e , i n p ar t, to

t h e r e l a ti v e l y s ma ll s iz e o f t he s e p ho s p h oa min o aci d s . H ow ever , en r i ch m en t

o f T y r ‐ph os p ho r yl at ed p r ot e i n s u s in g im mun o pr e ci p it at i o n can b e ver y

e f ﬁc i en t, e na b l in g t he id e nt iﬁ ca t io n o f h un d r ed s o f p h o s p h o r yla te d p r o tei n s

i n bi o l o g i ca l ti s s ue s ( R u s h et al ., 20 05 ) . A p ra ct i cal l im i tat io n wi th th i s

a pp r oa c h i s th a t n on t ar g et p r ot e in s ma y b e co ‐p u r iﬁ e d b y i n ter act i o n wi th

t h e ta r g e t pr o te i n s o r w it h t he an t ib o d ie s t he m s el ves , wh i ch , i n p r a cti ce , ar e

r a r e ly ( i f e v e r ) 1 0 0% s p eci ﬁ c f or t he t ar g et mo d i ﬁ cat io n . Fo r tu n at e l y, m an y

o f th e c omm e r ci a ll y av ai la bl e an t i ‐p ho s p ho t yr o s i n e an ti b o d i es ar e r eac t i ve

t o wa r d ph os ph oh is t id i ne (K l u mp p an d K r ie gl st ei n , 200 2 ) , wh i ch al s o co n -

t a ins a r e l a ti v e l y la r ge a r o mat ic s t r uc tu r e . S uch an ti b o d i es m ay th er e fo r e

b e e ff e cti v e i n r ec ov er in g b ot h Ty r ‐ an d H is ‐p h o s ph o r yl ate d p r o te in s ,

a l th ou g h th i s hy po t he s is h a s ye t t o b e t es t ed .

I mmo bi l i z ed me t al‐io n af ﬁ n i t y ch r o m at o gr a p h y ( I MA C) h as l o ng b een

u s ed fo r ta r g e te d e n r ich me nt o f p ho s p ho r y la t ed p r o tei n s ( A nd e r s s o n an d

Po r a th, 1 9 8 6 ) . T h is ap p li ca t io n ex p lo it s t he p ar ti cu l ar af ﬁn i ty o f p h o s ph a te

3

þ

3 þ

g r ou ps fo r me t a l io n s s u ch as Fe an d Ga , wh i ch ar i s es fr o m th e p r e-

d o mi na n tl y e l e c tr o s ta t ic in t e r ac ti on b et w ee n o xyg en at om an d tr i val en t

m eta l i on ( ne i t he r o f w hi ch is ea s il y p ol ar i zed ) . Fi gu r e 2 s h o ws th e s te ps

i nv o lv e d i n a ty p ic al I M AC ex t r a ct io n p r oc edu r e. Co m m er ci al k i ts b as ed

o n Fe ( I I I ) ‐ a nd Ga ( I I I ) ‐I M A C ar e av ai la bl e fo r s el ect ive en r i ch me n t o f

p h os p ho pr o te i ns f r om p r ot e i n ex t r act s ( W o ls c h in et al ., 200 5 ) . I m mu n o af ﬁ-

n ity ‐ba s e d ki ts for t he r em ov al o f h ig h ab u n d an ce p r o te in s ( H AP) ar e al s o

a v a i l ab l e , a nd c a n en h an ce d et ec t io n an d an al ys i s o f p h o s ph o r yl at ed an d

o t he r l ow a bu n d an c e p r ot e i n s ( LAP ) . M o st o f th es e co m m er ci al k i ts h ave

b ee n de v e l op e d a n d o pt im ize d f or u s e w it h m am m al ian ( e. g. , p l as ma )

p r o te i ns . Co ns e q ue n tl y , t he p r ot o co l s d ev el op e d fo r th es e p r o d u cts ar e

n o t a pp l i ca b l e to, o r ma y r eq u ir e ex t en s iv e m od i ﬁ cat io n fo r u s e wi th ,

5

54

genome‐wide analyses of two‐component systems

[27]

M

M = Immobilized metal-ion

=

M-chelating compounds

Other sample components

FIG . 2. I mmo b ili ze d me ta l‐io n a fﬁ n it y c h r o mat og ra p h y (I M AC). Se le c t ed m et a l io ns a re

l oad ed o n to a c hel atin g c o lu mn . Th e s am p le is t he n p as s ed t hr ou gh t he c olu mn , a nd c om -

p ou nd s wit h an afﬁ n it y f or t he immo b ili ze d me ta l io n s a re se le c t iv ely re t a ine d . O nc e t he

c o l u mn h as b een was h ed , t he r et a in ed c o mp o u nd s c an b e re c o ve r ed by di spl ac in g t he m f ro m

th e imm o bil ized meta l io n s . Th is c an b e a ch ie ve d b y e lu t ion wit h a c om pe t ing lig a nd or by

r ais i ng o r lo wer in g p H, d ep e n d in g o n t he n at u r e o f t he r et a ine d c om po unds .

p la nt a nd / or ba c te r ia l s ys t em s . M o re o ve r , r ec ove r y o f i n ta ct p h o s ph o p r o -

t ei ns us ing a fﬁni ty‐based metho ds r elies u po n t he s tabi li ty, and acces si bi lity,

o f t h e t ar ge t m o d iﬁ c a ti on . S uc h m e t ho ds m ay n o t , t h e r e f o r e, b e s u i ta b l e f o r

t he r ec ov er y of H is‐ph os pho rylated p rot ein s un less experi mental con di ti on s

can be a djusted t o p revent h ydr olys is o f t he pho sph oamid ate b on d d ur in g

e xtr ac ti on.

M etal oxi de a fﬁn it y chr omat ography ( MOAC) has been u sed s uccess ful ly

t o e nr ic h i ntac t pr ot eins cont ainin g p hos pho rylated Ser, T hr , and T yr r es idu es

(L aug es en e t a l., 2 006; W ols chin et al., 2 00 5) . Thi s techn iq ue com pares favor -

a bly with c omme rcial pr odu ct s f or ph os pho pr ot ei n enr ichm ent. Fur therm or e,

t he i nc uba ti on a nd elu tion buf fers r equired f or MOA C are non acidi c and

m ay , the re for e, pr es er ve p ho sph oh is tidin es , alt hou gh this techn iq ue h as yet to

be e va luated for t he r ecover y o f His‐p h os p hor yl at ed pr ot e in s.

Gel Separation

Fo ll o wi n g e x tr a ct io n , f r act i o na t io n , an d /o r p h o s p h o p r ot ei n en r i ch men t ,

i nd i v i du al pr o te i n s ca n b e f ur t h er r es o l ve d u s i ng o n e ‐ o r tw o ‐d i men s i o n al

g e l e l e c tr o ph or e s is ( 1 ‐ o r 2 ‐DE ) , d ep en d in g o n th e co m pl exi t y o f th e s am p l e

[

27]

identiﬁcation of histidine phosphorylations

555

o r fr a c ti on . Ge l e le c t r op h o r e s is al lo w s t he ex p r es s i o n o f i n di vi d u al p r o tei n s

t o be c omp a r e d am on g s am pl es an d d if fe r en t ia l ly exp r es s ed p r o tei n s to b e

t a r g et e d fo r i de n ti ﬁ ca t io n an d f ur t h er an al ys i s . T h i s r eq u i r es vi s u a l iz ati o n

o f th e g e l ‐s e p a r a ted p r o t ei ns u s in g au t or a d io gr ap h y, vi s i b le, o r ﬂ u o r es ce n t

3

2

s ta in in g me t ho ds . Au t or ad i og r ap h y o f P ‐l ab el ed p r o tei n s , fo l l o wi n g

s e pa r a ti o n by g el el ec tr o p h o r es is an d b lo t t in g o n to a p o ly ( vi ny l i d in e)

d iﬂ uo r id e ( PV DF) me m b r an e, is a w ell ‐es t a bli s h ed m et h o d fo r d ete cti n g

n ew ly s y nt he s i z e d p ho s p h o pr o t ei ns in s ys t em s th at ar e am en ab l e to m eta-

b o l i c r a di o la b el i n g, s u ch as ( b act e r ia l) ce ll cu l t u r es . T r eatm en t o f th e

PV DF me mb r a ne w it h b as e , p r io r t o au t o r adi o gr ap h y, h yd r o l y zes p h o s -

p h os e r i n e a nd ph o s p ho t h re o n in e b ut le av es p ho s p h o tyr o s i n e, p h o s ph o h is -

t idi n e , a nd ph os ph o lys i ne r es i d ue s in t ac t ( K lu m pp an d K r i egl s te in , 200 2 ) .

A d di ti o na l tr e a tm e n t w i t h ac id cl ea ve s t he p ho s p h o am id at e b o n ds , l eavi n g

o n l y ph os ph ot yr os in e r es i du es u nh y d r ol yz ed . Hen ce , o n e can d i s c r i mi n ate

a mo ng ph os ph opr o t ei ns co n t ai ni ng d if fe r en t t yp es o f p h o s p h o a m in o aci d s ,

a l th ou g h Hi s ‐ph os p h o r yl at ed p r ot e i n s t ha t al s o co n tai n p h o s ph o tyr o s i n e

r e si du e s ma y g o u nd e te ct ed u s in g t hi s ap p r o ach . H ow eve r , p r o tei n s ta r -

g e t e d us i n g a ut or a di o gr a ph y ma y n ee d t o b e e xci s ed fr o m r ep l i cate, n o n -

r a d i ol a be l e d e l e c t r op h o r e s is ge ls f or f ur t h er ( MS ) an al ys i s i n o r d er to

a d dr e s s s uc h i s s ues as s en s i t iv it y, r ec ov er y, an d h an d l in g o f r ad i o a cti ve

s a m pl e s ( Co nr a ds et al., 20 02 ) .

M on o c lo na l an t i b od i es ca n al s o b e u s ed t o d e te ct gel ‐s ep ar a ted p h o s -

p h op r ote i ns i n PV DF me mb r an e s . Ag ai n , t hi s We s te rn b l ot ti n g ap p r o ach

p r o v i de s i nfo r ma ti on ab o u t w hi ch t yp es o f r es i d u es ( e. g. , S er , T h r, o r T yr )

a r e ph os p ho r y l a ted in ea ch p r o t ei n, as ex em pl iﬁ ed i n Fi g. 3. U nf o r tu n at e l y,

t h e pr o du ct io n o f an t ib o di es s p eci ﬁ c f or p h o s p h o h is t id i n e i s h i nd e r e d

b y th e i ns ta b il it y o f t hi s mo d i ﬁ cat io n ( K l u m p p an d K r i egl s te i

H o we v e r , th e r e a ct iv it y o f ma ny an t i ‐p ho s p ho tyr o s i n e an ti b o d ie s to wa r d

F IG . 3. A s il ver‐s ta in ed 2‐DE p r o te in g el ( le ft ) a nd Wes t er n bl ot s ob t ai ne d f ro m re p lic a te

gels u s in g an tib o d ies a ga in s t p h o s p h o se ri n e ( mid d le ) a nd ph os ph ot h re oni ne re s idu e s (r ig ht ,

c o ur tes y o f Dr . Li an glu Wa n , N RC P lan t B io te ch n olo g y I ns t it ut e ). H ori zo nt a l sp ot t ra in s a re

c h ar ac ter is ti c o f mu lt ip le p h o s p h o ry lat io n , w hi ch h as a m uc h g re a t er e f fe c t on pI t ha n on

mo lec ular weigh t.

5

56

genome‐wide analyses of two‐component systems

[27]

p ho s p ho hi s ti d i ne s , w hi ch co u ld b e 10 t o 10 0 ti m es m or e ab u n d an t th at

p ho s p ho ty r o s i ne (M at t h ew s , 19 95 ) , ma y p r o vi d e a m ean s o f ta r ge ti n g

t h e s e pr o te i ns fo r s u b s eq u en t id e nt iﬁ ca t io n an d m ap p i n g o f p h o s p h o r yla -

t ion s i te s by ma s s s p ect r o met r y ( M S) ( s ee la t er ) .

W e s te r n bl o tt in g an d au t o r ad io gr a p hy ar e ver y s en s i ti ve te ch ni q u e s .

H e nc e , pr o te i n s d e t ect e d u s in g t he s e me th o d s m ay n o t b e o f s u fﬁ ci en t

a b un da n c e fo r i de nt iﬁ ca t io n an d s t r uc tu r a l a n al ys i s u s i ng MS , u n le s s a

n um be r of r e pl i ca t e ge ls ca n b e r u n an d cor r e s p o n d in g p r o tei n s p o ts

m a tc h e d a nd c omb in ed . I n co n tr a s t, p r ot e i n s d ete ct ed b y d i r ect i n ‐gel

m eth od s s uc h a s C oo m as s i e, s il ve r , o r ﬂ uo r e sce n t s ta in i n g can u s u al ly b e

i de n t i ﬁe d fo l lo wi n g ex ci s io n o f t he in d iv id u al b an d s o r s p o ts . Fu r th er m or e ,

t h e de v e l op me n t o f ﬂ uo r es c en t p r ob e s f or ce r tai n m od i ﬁ cati o n s ( e. g. , p h o s -

p h or y l a ti on , g l y co s yl at i on ) me an s t ha t ge ls can n o w b e s ta in ed o n ce fo r

d et e ct i on of th e t ar g et mo d iﬁ ca ti o n, an d ag ai n to vi s u a l iz e a n d co m pa r e

e x pr e s s i on l e v e l s f or ev er y p r ot e in in t he ge l ( Vy etr o go n et al . , 200 6 ) . T h is

a pp r o a c h do e s n ot p r o vi d e s it e ‐s p eci ﬁ c in f o rma t i o n an d i s , th er e fo r e, o f

l im i te d ut i li t y i n t ar g e t in g Hi s ‐p ho s p h or y la t ed p r o tei n s , u n le s s s u ch p r o -

t e ins ha v e be e n en r i ch ed p r io r t o ge l el ec t r o p ho r e s i s . S u ch i n fo r ma ti on

m ay , ho we v e r , be o bt a in e d b y co mp ar in g r ep li cat e gel s r u n b efo r e an d

a f t e r tr e a tm e nt w it h ac id , b a s e, o r s it e ‐s p eci ﬁ c p h o s p h ata s e s an d o b s er vi n g

ch a ng e s i n pr o te i n mi gr at io n an d /o r vi s u ali zat i o n th at ar e co n s is t en t wi th

d ep h os p ho r yl a ti on .

Mass Spectrometry

On ce th e pr o t ei ns o f in t er e s t h av e b ee n is o l ate d u s i ng fr a cti o n ati o n ,

a f ﬁn i ty , a nd / or g e l ‐b as e d ap p r o ach e s , o ne ca n us e m as s s p ect r o met r y ( MS )

t o i de n ti f y ta r g e t p r ot e in s an d s it e s o f mo d iﬁ cat io n ( S ch wep p e et al . , 200 3 ) .

Th e c onv e n ti o na l , ‘‘ b o tt o m ‐u p’ ’ ap p r oa ch t o p r o tei n MS i n vo l ves th e u s e o f

a si t e ‐s pe c iﬁ c pr o t ea s e, s u ch as t r yp s in , t o cl ea v e th e p r o tei n ( s ) i n to co m po -

n en t pe p ti de s . Pr ot ei n d ig es t io n , w hi ch n or m al l y ta ke s s eve r a l h o u r s , can b e

p er f or me d i n s ol ut io n ( u s i n g im mob i li ze d t r yp s i n b ead s , fo r exa mp l e) o r

i n ‐g e l , on c e th e s p ot s o r b a n ds o f in t e r es t h a ve b een r em o ved an d th e

p r ot e i n( s ) de s t a i ne d , r ed u ce d , an d al k yla t e d to exp o s e th e p r o teo l yti c

cl e a v a g e s i t e s . Aut o m at i on o f t hi s p r o ce s s s ig ni ﬁ can t l y r ed u ces co n tam i n a-

t io n ( e s pe c i a l ly fr om h um an k e r at in ) an d , t og et h er wi th au to m ate d gel s p o t

e xc i s io n, e na b l e s h ig h ‐t hr o u gh p u t p r ep ar a t io n an d d i ges ti o n o f p r o tei n

s a mp le s ( R os s e t al., 20 02 ) . Th e r es u lt in g p ep ti d es ar e am en ab l e to exa ct

m as s a n a l y s is us i n g a t ime ‐o f ‐ﬂ ig ht ( TOF ) o r F ou r i er tr a ns f o r m ‐i o n cyc l o -

t ro n r e s on a nc e ( FT ‐I C R) ma s s s p e ct r o met er eq u i p pe d wi th a m atr i x ‐

a s s i st e d l a s e r de s or p t io n / io n iz at io n ( M AL DI ) o r el ect r o s p r ay i o ni zat io n

(

ES I) s ou r ce ( A eb er s o ld a n d M an n , 20 03 ) . MA L D I an d ES I ar e m il d

[

27]

identiﬁcation of histidine phosphorylations

557

‘

‘d e so r pt i on ’ ’ i on i za ti o n t ech n iq u es t ha t ar e cap a b l e o f ge n er at in g s ta bl e,

i nt a c t mo l e c ul a r i o ns f r om p r o t ei ns an d p ep t ide s , fr o m wh i ch m as s an d / o r

s e q ue n ce i nfo r ma t io n ma y b e d er i v ed ( s ee f ol lo win g te xt) . U s in g a d ata b as e

s e ar c h e ng i n e s uc h as M S ‐Fit ( h tt p :// p r os p ec t o r .u c sf .e d u /) o r Ma s c o t ( h tt p: / /

www .ma t r i x s ci e n ce .co m/ ) , o ne ca n co mp ar e an d m at ch th e m eas u r ed

m as s e s of th e s e p ep t id es w it h t he o r et ic al ( t r yp ti c) p ep ti d e m as s es gen er -

a t ed i n s i l ic o fr o m a p r ot e in o r ge n e s eq u en ce d ata b as e s u ch as S wi s s Pr o t o r

N CBI n r . T hi s te c h ni q u e, k no w n as p e p ti d e ma s s ﬁ n g er p r i n ti n g ( PMF) , i s

g e ne r a l l y pe r fo r med u s in g a M ALD I ‐TO F mas s s p ect r om e te r , wh i ch p r o -

þ

v i de s r a pi d , s i mu l ta n eo u s an a ly s is o f p r ot o n at ed [M þ H ] m ol ecu l a r i o ns

g e ne r a te d fr o m th e p ep t id es in a p r ot e i n d ig es t (Y at es , 199 8 ) . PMF u s u al ly

wo r ks we l l i f th e s am pl e co n ta i n s o ne o r t wo ab u n d an t p r o tei n s an d th e

d at a ba s e s e a r ch i s w ell co n s tr ai n ed , f or ex am pl e , b y u s i ng d ata o f h i gh m as s

a ccu r a cy a n d/ o r s p eci fy in g t he o r ga ni s m( s ) u n d e r i n ves ti gat i on . Fu r th er -

m o r e , by i nc l ud ing p ho s p h or y la ti o n o f s p eci ﬁ c r es i d u es ( e. g. , S er / Th r ) as

v a r i ab l e mo di ﬁc a t io n s in t he s ea r c h , o ne ca n i d en ti f y p o s s i b l e s i tes o f

p h os p ho r y l a ti on , if p ep t i d es ca r r yin g t hi s mo d i ﬁca ti o n ap p ear as ad d i t i o na l,

s ig ni ﬁc a nt ma tc h es .

Ho w e v e r , to co n ﬁr m s it e s o f p ho s p h o r yl at io n an d i d en t i fy p h o s p h o r y-

l a t e d a n d ot he r pr o t ein s in co mp l ex mi xt ur e s (f o r exa mp l e, i n u n r es o l ved

p r ot e in fr a c ti on s o r ge l b a n ds ) , o ne mu s t r es o r t to ta nd e m m as s s p ect r o me -

t ry ( MS / MS ) . T his ap p r o ach t yp ic all y in v ol ve s liq u i d ch r o m at og r a p h ic ( L C)

s e p a r a ti on a nd on ‐li n e el ec t r os p r a y io n iz at i o n ( ES I ) , wh i ch gen e r at es m ul -

n þ

t ipl y pr o to na t e d [ M þ n H] mo l ec u la r io n s fr o m th e L C ‐s ep ar a t ed p ep -

t ide s . I nd i v i du a l p ep t id e ( p r ecu r s o r ) io n s ar e t hen s el ect ed an d fr a gme nt ed

u s in g a ta n d e m qu ad r u po le , q ua d r up o le ‐t ime o f ﬂ i gh t ( Q ‐T OF ) , i o n tr a pp -

i ng , or FT ‐I CR m as s s p ect r o me t er (Ae be r s o ld an d Ma n n , 200 3 ) . T h e fr a g-

m en t ( or pr o du ct ) io n s p ect r a ge n er a t ed b y th es e i n s tr u m en ts en co d es

i nfo r ma ti o n a b ou t t he am in o ac id s eq u en ce s o f th e s el ect ed p ep ti d es ,

i nc l ud in g mo di ﬁc a t io n s t o an y o f t he r es i d ue s . T h es e s p ect r a can b e d e co d ed

m an ua l l y , or by u s in g d e n ov o s eq u en c in g s o ft w ar e s u ch as PEA K S ( h tt p: / /

www .b io i nf or ma t i cs s o lu t io n s .co m/ ) , t o d et er mi n e th e s eq u e n ces o f th e p ep -

t ide s a nd a ny s i te s o f p ho s p h o r yl at io n , s in ce t hi s m od i ﬁ cat io n i n cr eas e s th e

m as s of a n a mi no ac id r es i du e b y 80 Da lt on s ( Da) . T h ey can al s o b e u s ed to

i de n ti fy th e pa r e nt p r ot e i n b y ma t ch in g t he ex p er i men t al MS / M S s p ect r a

wi th th e or e t i ca l ( t r y p ti c) p e p ti d e f r ag men t io ns gen e r at ed i n s i li co fr o m a

p r o te i n or g e ne s eq u en ce d at ab a s e, u s in g p r o gr am s s u ch as Ma s c o t o r

S eq u es t ( Y a te s , 19 98 ) .

T he pr o c e ss o f co ll is io n ‐in d u ced d is s o cia t ion ( CI D ) , co m m o n ly u s ed to

g e ne r a te d pe p ti d e f r ag men t s d ur i n g M S /M S , m ay r es u l t i n l o s s o f p h o s -

p h a te , de p e nd i ng o n t he r es i d ue a n d t he co ll is i o n en er g y u s ed . Fo r exa mp l e,

l os s of H PO ( 9 8 Da ) , w hi ch r es u l t s f r om ꢀ‐el i mi n ati o n o f th e p h o s ph a te

3

4

5

58

genome‐wide analyses of two‐component systems

[27]

g r o up , i s di a g n o s t ic f or p ep t i d es co n ta in in g p h o s ph o r yl ate d S er an d T h r

r e si du e s (Sc h we p p e et al ., 20 0 3 ) . I n co n t r as t , e xte ns i ve CI D r es u l ts i n th e

e l i mi n a ti on of ph o s p ho t yr o s in e as a s t ab l e p ho s p h o r yla te d i m m on i u m i o n

wi th a ma s s ‐to‐c har g e r at io ( m/ z) o f 21 6, w hi ch i s ch ar ac te r is t ic o f p ep ti d es

ca rr y i ng th i s mo diﬁ c at i o n ( S te en et al., 20 03 ) . Fi gu r e 4 s h o ws exa mp l es o f

p ro du c t‐i on M S/ M S s p ect r a o bt ai n ed f or s t a nd a r d p ep ti d e s co n tai n i ng

p h os p ho r y l a te d S e r , Th r , an d Ty r r es i d ue s u s ing a Q ‐T OF m as s s p ect r o me-

t e r. As i s a pp a r e n t f r om t he s e s p ect r a , f aci le lo s s o f p h o s p h ate fr o m S er an d

Th r te n d s to i nh i b it f ur t h er f r ag m en t at io n o f th e p ep ti d e, wh i ch , i n tu r n ,

m ay pr e v e n t l oca li za ti o n o f r es i du e s ca r r y i n g th i s m od i ﬁ cat io n . T h e

r e pl a c e me n t of p ho s p h at e gr o u p s w it h s t ab l e m ar k e r m ol ecu l es h as b een

u se d to e nh a nc e M S an d M S /M S an a ly s is , an d to en ab l e r el a ti ve q u a n ti -

ﬁ ca ti o n of ph os p ho r y la t ed p ep t id es an d p r ot ein s ( Co n r a d s et al . , 200 2;

W ol s c hi n e t a l ., 2 00 5 ) . Un f or t u n at e ly, s u ch t ech n i q u es ar e cu r r en t l y ap p l i-

ca bl e on l y to Se r an d Th r p ho s p ho r y la t io n s. N e ve r th el es s , l o s s o f p h o s p h ate

f ro m Se r or T hr du r in g M S /M S ca n b e u s ed t o tr i gge r s el ect io n an d fu r t h er

f ra g me n ta t io n of t he d e p ho s p h o r yl at ed p ep t id e wh en p er fo r m ed o n an i o n

t ra p pi ng or FT ‐I C R in s t r u men t ( B ea us o le il et al . , 20 04; G r uh l er et al ., 200 5 ) .

3

Th i s te c hn i qu e , k no w n as d a t a‐d ep en d en t MS / MS / MS ( o r MS ) , co u l d

2

9-Mar-2004

do032604_009 13 (14.823)

3: TOF MSMS 787.92ES+

3.11e4

1

476.8214

1

00

%

0

1574.8177

1

232.6765 1330.6515

7

39.0063

867.5016

1135.6608

110.0751

253.1067 366.1968

497.2859

1703.2194

do032604_008 1 (10.978)

3: TOF MSMS 792.85ES+

1.09e3

1

486.7209

1

00

%

0

1

584.6829

4

69.3239

1415.6573

1215.5702 1326.5938

7

43.8021

4

27.3015

939.4905

1

39.0918 235.1665

535.2751

856.4611

1086.5687

1679.6619 1777.6907

do032604_008 7 (15.292)

3: TOF MSMS 568.25ES+

1702.7756

472

1

00

%

0

629.3673

216.0488

486.2810

7

44.3699

8

45.4433

136.0775

373.2016

03.2231

3

568.2128

600

941.4189 1074.4512

900 1000 1100

1556.6837

1257.6064

1456.9760

1

778.0898

mass

100

200

300

400

500

700

800

1200 1300

1400

1500

1600

1700

1800

FIG . 4. Pr o d u c t‐io n MS /M S s p ec t r a f or S er ‐ a nd Th r‐ph os ph ory la te d pe p ti de s (u pp e r a nd

mi dd le p an els ) , in whi ch t he mo s t a bu n d a n t f r ag men t io n co rr e spon ds t o ne u tr a l lo ss of ph os -

p ho ri c ac id ( 98 Da) f r om t he in t ac t p ep t id e , a nd f or a Ty r‐ph os ph ory la te d pe p ti de (l owe r

p an el) , wh ic h gives th e d ia gn o st ic p h o s p h o tyr o s in e immo niu m io n (m /z 2 1 6) .

[

27]

identiﬁcation of histidine phosphorylations

559

b e us e d to de t e ct an d ma p s it e s o f p ho s p ho r y la ti o n i n H is ‐p h o s ph o r yl ate d

p ep ti de s , pr o v i de d t ha t in t a ct p ho s p ho p ep t id e p r ecu r s o r i o ns can b e

g e ne r a te d .

FT ‐I CR ma s s s p ect r o me t er s ar e al s o ca p ab l e o f p er fo r m in g exa ct m as s

M S a nd M S/ M S an a ly s is o n in t ac t p r ot e in s ( C oo p e r et al ., 200 5 ) , o b vi ati n g

t h e ne e d fo r pr o t eo l ys i s . Th is ‘‘ to p ‐d ow n ’’ ap pr o a ch al l o ws fo r a co m pa r i -

s o n of th e a c tua l ma s s o f a mo d iﬁ e d p r ot e in w it h th e th eo r e t i cal m as s o f th e

m atc h in g , un mo d iﬁ e d p r ot e in f ou n d in t he d at a b as e. Wh en co m bi n ed wi th

t h e M S/ M S da ta ge n er a t ed f or t he s am e p r ot e i n , th i s ap p r o ach p r o vi d e s a

p o we r fu l me th od f or id e nt if y in g an d lo ca t in g k n o wn an d n o v el m od i ﬁ ca-

t ion s i n ta r g e t pr o t ei n s . Fu r th e r mo r e, FT‐I C R i n s t r u me n ts ar e co m pa t i b le

wi th e l e ct r on c a p tu r e d is s o cia t io n ( E CD ) , a t ec h ni q u e th at p r es er v es l ab i l e

m o di ﬁc a t i o ns wh i le p r om ot in g f r ag m en t at io n an d s eq u en ci n g o f th e p ep -

t ide ba c kbo ne ( Emme t t, 2 00 3 ) . He nc e , FT ‐I C R MS an d EC D ap p ear we ll

s u ite d to th e i de nt iﬁ ca t io n an d s t r uc t ur a l an al ys i s o f p r o te in s co n tai n i n g

p h os p ho r y l a te d hi s t id i ne r es i du e s , al t ho u gh t he r eq u i r ed i n s tr u m en ta ti o n i s

v e r y e x pe n s i v e a n d n ot ye t w id ely av ai la bl e.

Phosphopeptide Enrichment

De s p it e th e i nh er e n t s en s i t iv it y a n d s p eci ﬁ ci ty o f m as s s p ect r om e tr y , th e

i de nti ﬁ ca ti on a nd s t r uc t ur a l a n al ys is o f p ho s p h or y lat ed p ep ti d es b y MS an d

M S /M S c a n s ti l l b e p r ob le mat ic , s in ce p ho s p ho r yl a ti o n i s u s u al l y s u b s to i -

ch io me tr i c a nd t en d s t o in h ib it p os it iv e io n i zat io n b y MA L D I o r ES I

(

a pr e r e qu is i te fo r p e p ti d e f r ag m en t at io n an d s eq u en ci n g) . Fo r th i s r eas o n ,

r e se a r c he r s c ont in u e t o in v es t iga t e w ay s o f en r i ch in g p h o s ph o r yl ate d p ep -

t id e s fr o m pr o te i n d ig es t s p r io r t o M S an a ly si s . S tr o n g cat io n exc h an ge

(

S CX ) c hr om a to gr a p h y is o ft e n u s e d as t he ﬁ r s t s te p i n m ul ti d i m en s i o n a l

l iq ui d c hr om a to g r a p hy ( M D LC ) , an al t er n at i ve ap p r o ach to gel ‐b as ed

p r o te i n a n a l y s is ( W o lt er s et al ., 20 0 1 ) . E nt ir e pr o t ein ext r acts o r fr a c ti o n s

a r e di g e s te d , us u a ll y w it h t r yp s in , an d t he r es u lt i n g p ep ti d es l o ad ed o n to a

S C X c ol um n . So lu t io n s o f in cr e as i n g s al t co n c en tr at io n ar e u s ed to el u te

p ep ti de fr a c ti on s f r om t he co lu m n , w hi ch ar e t h en s ep ar at ed b y r eve r s ed ‐

p h a s e hi g h pe r f or ma nc e li q ui d ch r o ma to gr a ph y ( R P ‐H PLC ) an d an al yz ed

o n ‐l i ne by ES I ‐MS /M S , o r o ff ‐li n e b y M ALDI ‐MS / M S . A s wi th p r o tei n s ,

f ra c ti on a ti on of p ep t i d es ca n as s i s t in t he d e t ect io n o f p h o s ph o r yl ati o n s i tes

b y r e du ci ng s a mpl e co mp le x it y d ur i n g s u b s eq u e n t an al yt i cal s te ps ( G r uh l er

et a l . , 2 0 0 5 ) . Fu r the r mo r e, w he r ea s u nm od i ﬁ ed tr y pt ic p ep ti d es ( wh i ch h ave

b a s i c C‐te r mi n a l r es i d ue s ) u s ua ll y ca r r y a n et ch ar ge o r 2 þ o r gr ea t er i n

s o lu ti on , th e pr e s e nc e o f a n eg at iv el y ch ar g ed p h o s p h ate gr o u p gen e r al l y

l im i ts th i s c ha r g e t o 1 þ. C on s e q ue n tl y, mo s t p ho s p h o p ep ti d es te nd to el u te

f ro m th e SC X c olu mn b ef o r e u np h o s p ho r yl at ed p ep ti d es . T h is ap p r o ach

5

60

genome‐wide analyses of two‐component systems

[27]

h a s be e n us e d s ucc es s f ul ly t o en r i ch p ho s p h or y lat ed p e p ti d es fo r o n ‐l in e

M S a na l y s i s , g r e a t ly r ed u ci ng b ac kg r ou n d in t e r fer e n ce/ i on s u p p r es s i o n b y

u n ph os ph or y l a te d p ep t id es ( B ea us o le il et al . , 200 4 ) . Wh e n co m bi n ed wi th

s ta b le i s oto pe l a be lin g t ech n iq u es s u ch as S I L A C ( G r uh l er et al . , 200 5 ) ,

t h i s g e l ‐fr e e a pp ro a ch ca n al s o b e u s e d f or r el a ti ve q u a n ti ﬁ cati o n o f p h o s -

p h op r ot e i ns i n di ff er e n t s am pl es . Un f or t u na t el y, H is ‐p h o s p h o r yl ated p ep -

t ide s a r e un l i ke l y t o s u r vi ve t he ac id i c co n d it io n s n e ces s a r y fo r S CX o r

r e v e r s e d ‐ph a s e l i q u id ch r o m at o gr a p h y.

St r on g a ni o n e xc h an ge ( S AX ) ch r o m at o gr a p h y h a s al s o b e en eva lu a te d

f o r pe p ti de fr a c ti o n a t io n (N u¨ h s e et al., 20 03 ) p r i or to en r i ch men t o f p h o s -

p h op e pt i de s us i ng I M AC. Th e la t te r , w hi ch is b as ed u p o n th e af ﬁn i ty o f

o xy g e n ‐c on ta i ni n g gr o u p s ( e. g., p ho s p h at e , ca r bo xy lat e) fo r tr i val en t m etal s

i on s , c a n be ma d e mo r e s p eci ﬁ c f or p ho s p h o pe pt i d es b y co n ver t i n g car b o x-

y l i c a c id g r ou ps to me t h yl es t e rs (Fic ar r o et al ., 200 2 ) . H ow eve r , th i s p r o ces s

b ia s e s a g a i ns t I MA C r ec ov er y o f s in g ly p ho s p h o r yla te d p e p ti d es an d m ay

l e a d to un wa n te d p ep t id e mo d iﬁ ca t io ns ( W o ls ch i n et al . , 200 5 ) , m ak in g i t

u n s ui t a bl e fo r ph o s ph o h is t id in es . S AX h el ps t o co m pe n s at e fo r th i s b i as b y

p er m it t i ng di ff e r e n t ia l el u t io n o f s in g l y an d mu l ti p l y p h o s ph o r yl ate d p ep -

t ide s us i n g di ff e r e n t s al t co n cen t r at io n s ( N u¨ h s e et al . , 200 3 ) . U nf o r tu n at el y,

t h e r e co v e r y of H i s‐p ho s p ho r y la t ed p ep t i d es fr o m S AX fr a c ti o n s i s al s o

co mp r om i s e d by t he ac id ic co n d it io n s r eq u ir e d fo r d es al ti n g b y s o l id ‐p h a s e

e x tr a c ti on ( SP E) p r io r t o I M A C an d /o r M S an al ys i s .

I MA C ha s be en u s ed ex t en s iv el y f or s el ec tive r eco ve r y an d en r i ch men t

o f pe p ti de s c ont ai n in g p ho s p h o r yl at ed S e r , Th r , an d T yr r es i d u es fr o m

p r ot e i n di g e s ts . A s w it h in t a ct p ho s p ho p r o te in s , th e m et al i o ns m os t co m -

3

þ

3 þ

m on l y us e d fo r thi s p ur p o s e ar e Fe an d Ga ( N u¨ h s e et al ., 200 3; Po s ew i tz

a nd T e mp st , 1 9 99 ) . I M A C me di a co n s is t o f a ci di c, m et al ‐ch el a ti n g fu n c-

t ion a l g r ou p s bo un d t o a s o lid s u p p or t s u ch a s S e p h ar o s e, aga r o s e , cel l u l o s e,

p o l y s t y r e ne r e s i n, o r s il ic a ( Li u et a l., 20 04 ) . T h e m os t co m mo n l y u s ed

f u nc t i on a l g r ou p is im in o d iac et at e ( I D A) , w hi ch i s s m all , h y d r o p hi l ic , an d

b in ds me t a l i on s t ig ht ly w hi le le av in g co o r d i na ti o n s i tes ava i la b le fo r

p ep ti de or pr o te i n b in d i n g ( Ar n o ld , 19 91 ) . Th e m os t p o p ul ar al te r na t i ve,

n itr i l o tr i a c e ta te ( N TA ) , s u r r ou n d s an d b in d s m etal s m or e ti gh tl y th at I D A ,

a l l owi n g s tr o ng me ta l ch el a t or s ( e. g., Hi s ‐t ag ged p r o tei n s ) to b e r eco ve r ed

b y I MA C wi th ou t s t r ip p in g t he me ta l f r om t he co l u m n . S eve r al p r o d uc ts

a r e c omm e rc i a l ly av ai la bl e f or p ep t i d e p ur i ﬁc at io n b y I MA C, i n clu d i ng

co l um n pa c k in g ma t er i al s , p r ep ac ke d an a ly tic al o r ext r acti o n co l u mn s ,

a nd pr e l oa d e d e xt r ac ti o n b ea ds . Di sp o s ab le p i pe t te ti p s p ack ed wi th

I MA C me d i a ha v e al s o b ee n d ev el op e d , w hi ch al l o w r ap i d en r i c h me n t o f

p h os p ho pe p ti de s f r om s ma l l vo l um es o f p r ot ei n d i ges ts . T h es e ti p s m ak e

i t po s s i bl e to e x tr a ct Hi s‐p ho s p ho r y la t ed p ep t i d es wi th s u fﬁ ci en t s p eed to

p re s e r v e th e i nta c t p ho s p h o h is t id in e r es i d u e f o r s u b s eq u en t i d en ti ﬁ ca ti o n

[

27]

identiﬁcation of histidine phosphorylations

561

b y M S ( N a pp e r et al., 20 03 ) . Fu r th e r mo r e, it i s p o s s ib l e to d i s cr im i n ate

b et we e n pe p ti de s co n ta in in g p ho s p h oh i s ti di ne an d p h o s p h o es te rs u s i ng

I MA C ( s e e fo l lo w in g t ex t) , s o met h in g t ha t ca n n o t b e ach i e ved u s i ng S CX ,

S A X, or M OA C, al t ho u gh t he la t te r is ef f e ct iv e i n en r i c h i ng p h o s ph o r yl ate d

p ep ti de s a s we l l as in t a ct p ho s p ho p r o t ei ns ( W ol s ch i n et a l. , 200 5 ) .

Identification of Phosphohistidine in a Model Protein

Ha v i n g r e v i ew e d t he t ech n iq u es cu r r en t ly ava il ab l e fo r ta r ge ti n g an d

a n a l y zi n g ph os p h o r yl at ed p r ot e in s an d p ep t i d es , i t i s ap p a r en t th at o n ly

r a p i d a nd / or no n i n va s ive me th o d s ar e li k ely t o b e ef fe cti ve i n r eco ve r i n g

i nt a c t Hi s ‐ph os p h o r yl at ed p r o t ei ns , an d id e nt if yi n g p h o s ph o h is t id i n es wi th -

i n th e s e pr o te i n s . Th e f ol lo w in g d e s cr i be s a p re li m in ar y i n ves ti ga ti on i n to

t h e us e of tw o s u ch me th o ds , I M A C an d M AL D I ‐T OF MS , fo r s el ect ive

r e co v e r y a nd i de nt iﬁ ca t io n o f a Hi s ‐p ho s p ho r y la te d p ep ti d e d er i ved fr o m

t h e ph os p h oc a r r i er p r ot e in HPr in E s ch er i ch ia co l i . H Pr o ccu p i es a cen t r al

r o le i n th e ba c te r ia l PTS , w hi ch me di at e s p ho s ph o r yl a ti o n ‐d e p en d en t s u gar

u p ta k e ( M ea d ow et al . , 19 90 ) as w ell as n ume r ou s r egu l at or y r o l es i n

b ac te r i a l me ta b ol i sm ( Po s tm a et al ., 19 93 ; Ti tge m eyer , 199 3 ) . H Pr u n d e r -

g oe s ph os p ho r y l at io n at a co n s er v ed h is t id i ne r es i d u e ( H is ‐15) an d fu n c-

t ion s a s a ph os ph ot r an s f er p r o t ei n b et w ee n E nzy m e I , th e i n i ti at in g en zym e

o f th e PT S, a n d a s u g ar‐s p eci ﬁ c E nz yme I I A p r o tei n ( A nd e r s o n et al. ,

1 9 7 1 ; W a y g oo d e t al . , 19 85 ) . Th e k in et ic s o f H Pr p h o s p h o r yla ti o n an d th e

p h os p ho hy d r ol y s is p r op er t ie s o f t hi s p r o t ei n ar e we ll es t ab l i s h ed .

Phosphorylation and Digestion of HPr

g l c

En zy me I , En zy me I I A , an d HPr p r o t ei ns fr o m E. co l i we re p u r iﬁ e d to

h o mo g e ne i ty us i ng p r ev io u s ly p ub l is h ed p r o to co l s ( A nd e r s o n et al. , 199 1;

Br o kx e t a l., 2 0 00 ; N ap p er et al ., 20 01 ) , as wa s th e Ch eY p r o tei n fr o m

S al m on el l a t y p hi mu r iu m ( S to ck et al . , 198 5) . Ph o s p h o r yl ati o n o f

a ch i e v e d by c omb in in g 2 0 n g o f E nz y me I w it h 10 ꢁg ( 1. 1 n mo l ) o f H Pr i n

2

p h oe n o l py r u va t e, an d 20 m M HE PE S b uf f er ( pH 7. 0) . Co m p l ete p h o s ph o r -

0 ꢁl v o l ume s of a r ea ct io n mi xt u r e co n ta i n in g 2 m M Mg Cl , 5 mM p h o s -

2

ꢁ

y l a ti on of HPr w as ac h ie ve d af t e r in cu b at io n at 37 fo r 15 m in . Fo r

u n ph os p ho r y l at e d co n tr o ls , t he p ho s p h o d on o r ( p h o s ph o en o l p yr u vat e) wa s

o m i tte d fr o m th e mi xt ur e .

R a pi d pr o te ol ys i s w as ac h ie ve d b y ad d i n g 2. 5 ꢁl o f 0. 4 m g/ m l

S ta p hl oc o cc us a ur eu s V 8 p r o t ea s e ( en d o pr o t ei n as e C) i n 0. 1 M b i cin e

(

p H 8 .6 ) a nd 1 m M E DT A t o a 20 ꢁl vo l um e o f p h o s ph o r yl ati o n r eac ti on

ꢁ

m ix tu r e . Di g e s t s w er e in cu b at ed at 37 f or ju s t 30 m in i n an at te m pt to

p r es e r v e th e ph os p h oh i s ti di n e r es i du e s , w hi ch un d e r go ext en s i ve h yd r o l y s i s

5

62

genome‐wide analyses of two‐component systems

[27]

d ur i n g l on g e r di g e s t io n p er i od s (An d er s o n et a l. , 199 3 ) an d ar e th er e f o r e

i nc om pa t i bl e wi t h co n ve nt io n al t r yp s in d ig es t i o n p r o ced u r es . T o es t im a te

t h e ﬁn a l pe p ti de co n ce n tr a ti o n, al iq u o ts o f t he V 8 d i ges t we re s ep ar a ted

b y SD S ‐PAG E an d t he in t en s it ie s o f d ig es t ed ( p ep ti d e) an d u n d ige s te d

(

pr o te i n) ba n d s q ua n t iﬁ ed u s in g a p ho s p h o im ager . T h e i n ten s i ty r at io o f

d ig e s te d to un di g es t ed b an d s w as 10 :1 , co r r es po n d i n g to a m axi m um th eo -

r e ti c a l c onc e nt r at io n o f 4 4 p mo l/ ꢁl f or t he p ho s p h o r yl ated H Pr p e p ti d e

V TI T A PN GL ( pH ) TR P AAQF V K E ( r es i du e s 6– 25) .

IMAC Conditions

Di s po s a bl e me ta l ‐ch el at i n g p ip et t e t ip s (Z ip T i p _M_C) , o b tai n ed fr o m

M il l i po r e Co r po r at i on ( B ed fo r d , M A) , w er e u s ed to fa cil i ta te r ap i d e xt r ac-

t ion a nd e nr i c hme n t o f p ho s p h o r yl at ed p e p tid es . T h e ti p s we re wa sh e d

1

0 ti me s wi th 1 0 ꢁl o f 0. 1% ac et ic ac id in d e io n i zed wa t er , th en ch ar ge d

wi th me t a l i on s by as p i r at in g an d d is p en s in g 1 0 ꢁl o f a 100 m M m et al s al t

s ol u ti on 1 5 ti me s . S ev er a l me t al io n s w er e eva lu a te d fo r th ei r ab i l it y to

s e l e cti v e l y e x tr a c t an d r ec ov er Hi s‐p ho s p ho r yla te d p ep ti d es b y I MA C,

3

þ

2 þ

3 þ

i nc l ud i ng Ga , C u , a n d F e . Th e ch ar g e d ti p s we r e r i n s ed 5 ti m es

wi th 1 0 ꢁl of de i o ni zed w at er , an d 5 t ime s w it h 10 ꢁl o f 0. 1% ace ti c aci d

i n 5 0 % a c et on i tr il e. Ph o s p ho p e p ti d es w er e lo a d ed o n to th e ti p s b y as p i r at-

i ng a nd di s p e ns i ng 10 ꢁl o f s am pl e 10 t ime s , th en wa s h i ng 10 ti m es wi th

1

i n 2 ꢁl of 1% NH OH an d im med i at e ly n eu tr a li zed wi th 1 ꢁl o f 2% tr i -

0 ꢁl of 0 .1 % a c et ic ac id in 50 % ac et o ni tr il e. Ph o s p h o pe p ti d es we r e el u ted

4

ﬂ u or o a c et ic a c id ( TFA) . S amp l es w er e t he n mi xed wi th an eq u al vo l u me o f

ꢂ‐c y an o ‐4 ‐hy d r ox yci n na mic ac id ( C HCA) ma tr i x s o l ut io n ( 5 m g/ m l i n 75%

a c et on i tr i l e wi th 0. 1 % TFA ) an d a p pl ie d d ir ect ly to th e MA L D I ta r ge t

p la te . Fo r c omp ar is o n , t he p ho s p ho r y la t ed HPr d i ges t wa s d es al te d b y S PE

u si n g c on v e nt io n al C₁₈Z ip Tip s ( M i ll ip o r e) , acc or d i n g to th e m an u f ac-

t u r e r ’ s i ns tr u ct i o ns , t he n co mb i n ed w it h an eq u al vo l u m e o f th e s am e

M AL D I ma tr i x so l ut io n , w i t ho u t p er f o r mi ng IM A C.

MALDI‐TOF MS Conditions

Ph os ph o pe p ti d es w er e an a ly z ed b y M ALD I ‐T OF MS u s i ng a V o yag er

D E ‐ST R i ns t r um en t ( Pe rs e p ti v e B io s ys t em s , F r ami n gh am , MA ) o p er ati n g

i n th e l i ne a r , r e ﬂe ct r o n , o r p os t ‐s o ur c e d ec ay ( PS D) m od e s wi th p o s i ti ve o r

n eg a t i v e i on i za ti o n ( N ap p er et al . , 20 03 ) . R eﬂ e c tr o n T OF p r o vi d e s th e h i gh

m as s r e s ol ut i on n ec es s ar y f or ex ac t ma s s me as u r em en ts , wh er eas l in ear

TO F pr o v i de s g r ea t er s en s i t iv it y at t he ex p en s e o f r es o l u ti o n . PS D i s a

d is s o c ia t io n pr o c es s t ha t p r ov id e s p ep t id e f r ag m en t i n fo r m ati o n ( al b ei t at

l ow ma s s r e s ol u ti o n ) f r om w hi ch s it e s o f p ho s p h o r yl ati o n m ay b e i n fe r r ed .

[

27]

identiﬁcation of histidine phosphorylations

563

Fu l l s ca n s pe c tr a w er e o bt a i n ed b y co mb in in g an d p r o ces s i n g 100 s can s , a

p r oc e s s th a t ta k e s le s s t ha n a mi nu t e. Fo r PS D an al ys i s , th e r eﬂ ect r o n

m i rr o r r a ti o wa s ad j us t ed in cr e men t al ly , t he r es u l ti n g s p ect r a co m bi n ed ,

a n d fr a g me n t i on p e ak s as s i gn e d u s in g t he in s t r u m en t s o ftw ar e ( a p r o ced u r e

ca n be pe r fo r me d au t om at ica l ly , an d mo r e r ap i d l y, o n n ewe r MA L D I ‐T OF

i ns tr u m e nt s ).

Enrichment of His‐Phosphorylated Peptides

Po s it i v e‐i on re ﬂ ect r o n M ALDI ‐TO F M S an al ys i s o f th e V 8 d i ges t o f

p h os p ho r y l a te d HPr ge n er a t ed a s p ect r u m co n tai n i n g m os t o f th e exp e ct ed

p ep ti de s , a s s ho w n in Fig . 5A . Th e Hi s ‐co n tai n i ng p e p ti d e ( 6–2 5) wa s

A

1

00

474.6

90

80

70

60

50

40

30

20

10

0

m/z 2150

6−25)

(

1

718.5957

0

4000

7

00

1360

2020

2680

3340

m/z 2150

(6−25)

^B1

00

3762.0

9

8

7

6

5

4

3

2

1

0

4000

7

00

1360

2020

2680

3340

F IG . 5. Po s i tive‐io n r eﬂ e c tr on MAL DI‐TOF MS s p ec t r a of (A ) a c om plet e V 8 di ge s t of t he

Hi s‐p h o sp h o rylat ed p r o te in HP r , a nd ( B ) a C u( I I )‐I MAC e x tr a ct of t he sa m e di ge s t. T he H is‐

c o ntain in g p ep tid e ( 6–2 5) is s el ec ti ve ly r ec o v er e d , b u t d et e c te d a lmo st e x clu siv e ly in no np ho s-

p ho ry lated fo r m ( m/z 2 15 0) . Th e e xp e ct ed p o s it io n o f t he p ho sp ho ry lat e d pe p ti de (m /z 2 2 30 ) is

i nd ic ated b y

_▾.

5

64

genome‐wide analyses of two‐component systems

[27]

d e te c t e d i n un ph o s p h or y la te d f or m ( m/ z 21 50 ) ; h o wev e r , th e p h o s p h o r y-

l a te d fo r m ( m/ z 22 30 ) co u ld n ot b e o bs er v e d , i n di cat in g th at h i s ti di n e

p h os p ho r y l a ti on is in co mp le t e, la b ile , an d /o r i n hi b i ts i o ni zat io n u n d e r

e x pe r i me n ta l c on d it io n s . An al ys i s o f t he C u( I I ) ‐I MA C ext r a ct o f th e p h o s -

p h or y l a te d HPr d ig es t co n ta in ed a s in g le in te ns e p ea k at m / z 21 50, as

s ho wn i n Fi g . 5 B. Ag ain , t hi s co r r es p on d s t o th e p r e d i cted m as s o f th e

H i s ‐ph os p ho r y l a t ed p ep t i d e w it ho u t t he p ho s p h ate gr o u p ( H PO ) . Wh e n

3

t h e s a me pr o to co l w as ap p li ed t o a V 8 d ig es t o f u n p ho s p h o r yl at ed H Pr , n o

p ep ti de i on s we r e d et ec te d , ev en w he n o pe r ati n g i n th e m or e s en s i ti ve

l in e a r mo de . T hi s s u gg es t s t ha t C u( I I ) ‐I M AC ext r acti o n i s s el ect ive fo r th e

p h os p ho r y l a te d f or m o f t he Hi s ‐co n t ai ni ng p ep ti d e, b u t th at th e p h o s p h ate

g r ou p i s l os t be f o r e o r d ur i n g M ALD I‐TO F M S an al ys i s .

Selectivity for Phosphorylated Histidine

2

þ

Of th e me ta l io n s in v es t i ga t ed , o nl y C u p r o ved ef fe cti ve i n r et ain -

i ng a nd r e co v e r i n g p ep t i d es t ha t co n ta in p ho s p h o hi s t i d in e r es i d u es . T o

v e r i fy th a t pe p ti d es co n ta in in g u np h o s p ho r yla ted h i s ti di n e r es i d u es ar e

n o t r e co v e r e d us i ng t hi s p r oc ed u r e , a mi xt u r e co n tai n i ng fo u r p u r iﬁ e d

g l c

p ro te i ns ( En z y me I , E nz yme I I A , an d HPr fr o m E. co l i , an d Ch eY fr o m

S al m o ne l l a t y ph im u r iu m ) , ea ch at a co n cen t r at io n o f 1 m g/ ml , we re d i ges te d

t o c omp l e ti on wi t h V 8 p r ot e as e an d s u b je ct ed to th e Cu ( I I ) ‐I MA C ext r a c-

t ion pr o to co l . T his mi xt ur e co n t ai ns 11 u ni q ue p ep ti d es b ear i n g H i s r es i -

d u e s ; ho we v e r , n on e o f t he s e w as r ec ov er ed u s i ng th e Cu ( I I ) ‐I MA C

p ro ce d u r e .

Di v a le n t tr a n s it io n me t al io n s ar e k no w n to i n te r act s tr o n g l y wi th

2

þ

u nm od i ﬁe d Hi s r es i du e s ; f or ex am pl e, N i –N T A i s u s ed r o u ti n el y fo r

p u r i fy i ng r e co mbi na n t p r o t ei ns in w hi ch a t er m in a l p o ly ( h is t i d in e) ta g h as

2

þ

b ee n i nc or po r a te d. M o re o ve r , C u –I D A h as a h i gh er af ﬁn i ty fo r H i s ‐

2

þ

co nt a i ni n g pr o te i n s t ha n d oe s N i –I D A ( Ar n o ld , 199 1 ) . Wh y , th en , d o e s

Cu ( II ) ‐I MA C not r ec ov er p e p ti d es co n ta in in g u n mo d i ﬁ ed h i s ti di n e u s i ng

t h e pr o to co l de s cr i b ed ab o ve ? To an s w er t hi s qu e s ti o n , we n eed to co n s i d er

t h e a c id /b a s e pr op e r t ies o f h is t id i n e an d t he p H o f th e s am p l e d u r i n g

d iff e r e nt s te p s i n t he I M AC p r oc es s . Th e ex t r a cti o n o f H is ‐ta gged p r o tei n s

b y I MA C i s ba s e d o n t he in t e r ac ti on b et w ee n d i val en t m etal i o ns an d th e

i mi da z ol e g r ou p o f h is t id in e. Un d er b a s ic co n d i ti o n s, th e u n ch ar ge d i mi d -

a z ol e r i ng fo r ms a co mp le x w it h t he im mo b ili ze d m etal i o n. El u ti o n o f H is ‐

t a gg e d pr o te i ns an d p e p ti d es is t he n ac h ie ve d b y l o wer i n g p H an d p r o to -

n at i n g th e i m i da z o le n it r og en ( p K 6. 0) , w hi ch gen e r at es a p o s it iv e ch ar ge

a

t h a t i s r e pe l l e d b y t he im mo b ili zed me ta l ca t io n . D ur i n g th e af or e m en -

t ion e d Cu ( I I ) ‐I MA C p r oc ed u r e, t he b in d in g o f u n p ho s p h o r yla ted

h is ti d i ne s i s pr e v en t ed b y lo a di n g t he s am pl e u n d e r th e aci d i c co n d i ti o n s

[

27]

identiﬁcation of histidine phosphorylations

565

t h at r e s ul t fr o m m ix in g o f t he w ea kl y b uf f er ed s am p l e wi th r es i d u al ace ti c

a ci d on th e I MA C co lu mn . Th is r ed u ce s t he s amp l e p H fr o m an i n it ia l val u e

o f 7 .0 ( 2 0 mM HE PE S) t o ar o u n d 3. 5 ( 0. 1 % ace ti c aci d ) , at wh i ch p o in t

p r o to na t i on of the im id a zo le r in g n eg at e s in t e r act io n wi th th e i mm o b i li zed

2

þ

Cu i on s . Su bs e q ue n t w as h in g w it h 0. 1% ac et ic i n 5 0% ace to n i tr i l e s er ve s

t o ma i nt a i n th e im id a zo le in p r ot o n at ed f or m , al th o u gh th i s m ay al s o

h yd r ol y z e s om e of t he in t ac t p ho s p ho p ep t id es ( s ee fo l l o w i n g te xt) .

Detection of His‐Phosphorylated Peptides

Pr e v i ou s s tu d ies h av e s h o wn t ha t s w it c h in g fr o m th e p o s it iv e to th e

n e g a ti v e i on i z a ti o n mo d e in cr ea s es M S d et ec ti o n s en s i ti vi ty fo r p h o s p h o -

r y la t ed pe p ti d e s r el at iv e t o t he i r u np h o s p ho r yl at ed co u n te r pa r ts ( Jan e k

m/z 2148

(

6−25)

^A¹00

1156.3

9

8

7

6

5

4

3

2

1

0

m/z 2228

+HPO₃)

(

m/z 2130

−H O)

(

2

0

2500

2

000

2100

2200

2300

2400

m/z 2132

−H O)

(

^B¹00

2

2713.9

9

8

7

6

5

4

3

2

1

0

m/z 2150

6−25)

(

m/z 2230

+HPO₃)

(

0

2

0

2500

000

2100

2200

2300

2400

F IG . 6. M ALDI m as s s p ec t r a o b ta in e d u s in g ( A) n eg a tiv e‐io n lin ea r T OF M S a na ly sis of t he

Cu ( I I )‐I M AC extr ac t f r om a V 8 p r o te o ly tic d ig es t o f p h o s ph or yl at e d H Pr , a nd (B ) po st‐so ur c e

d eca y ( PS D) o f th e His‐p h o s p h o ry lat ed HP r p ep t id e ( m/z 2 2 30 ) g e ne ra t ed in po sit iv e‐io n m ode ,

b ot h o f wh ic h c on ﬁ r m r ec o v er y o f t he in t ac t p h o s p h o p ep tid e.

5

66

genome‐wide analyses of two‐component systems

[27]

et a l., 2 0 0 1 ) . As ex p ec te d , n eg at iv e ‐io n M ALD I ‐T OF MS an al ys i s o f th e

Cu ( II ) ‐I MA C e xt r a ct d et ec t ed an io n o f m/ z 222 8, co r r es p o n d in g to th e

d ep r o to na t ed [M – H] mo le cu l ar io n o f t he i n ta ct H Pr p h o s ph o p ep ti d e,

ꢂ

a s s ho wn i n Fi g . 6A . Ho w ev er , t he u np h o s p h or yl at ed fo r m o f th i s p ep ti d e

(

m / z 2 1 4 8 ) wa s a l s o o bs e r ve d in t he s p ect r u m. Th i s fu r t h er s u gge s t s th at th e

i nta c t Hi s ‐ph os p h or y la te d p e p ti d e is r ec ov er ed u s i ng th e Cu ( I I ) ‐I MA C

p ro ce d u r e bu t i s s u s cep t ib l e t o d ep h o s ph o r yl at io n d u r in g I MA C an d / or

M S a n a l y s is .

T he s a me e x tr a ct w as s u b s eq u en t ly an a ly ze d i n p o s i ti ve‐i o n m od e u s i ng

PS D wi th th e i on ga t e s et at m/ z 22 30 , t he exp ect ed val u e fo r th e i n tact

p h os p ho pe p ti de . Th e r es u lt i n g s p ect r u m, s h ow n i n Fi g. 6B , co n tai n s i o ns

t h a t c or r e s po nd to s eq u en t i al lo s s o f HPO ( 80 D a) an d H O ( 18 D a) fr o m

3

2

t h e Hi s ‐ph os ph or y la t ed p ep t id e, w hi ch is al s o o b s er ved i n th e s p ect r u m.

S imi l a r r e s ul ts h a ve b ee n r ep o r t ed f or col l i s i o n ‐i n du ced d i s so c ia ti o n

(

CI D) of Hi s ‐ph os p ho r y la t ed p ep t id e io n s g en er a ted b y MA L DI ( Jan e k

et a l., 2 0 0 1 ) , c onﬁ r m in g t ha t t he in t ac t p ho s p h o pe p ti d e wa s r eco ve r ed b y

Cu ( II ) ‐I MA C. Fig u r e 7 s h ow s t he d if f er e n t s t r u ctu r e s o f p h o s p h o es ter an d

p h os p ho hi s ti d i ne r es i d ue s t ha t gi ve r is e t o n e u tr al l o s s es o f 98 an d 80 D a,

r e s pe c ti v e l y .

O

OH

OC

C

P

−

H PO (98 Da)

3 4

HO

O

CH

C

H₂

H C

2

HN

Dehydroalanine

Phosphoserine

O

OH

P

HO

N

HN

OC

CH

OC

CH

−

HPO₃(80 Da)

N

C

H₂

C

H₂

HN

Phosphohistidine

Histidine

3 4 3

FIG . 7. Ch ar ac teris t ic lo s s o f H P O a nd HP O f r om, re s pe c t ive ly , ph os ph os er ine a nd

p ho sp h o h is tid in e r es i d ue s . Th e f or m er ( w hi ch a ls o a pp l ies t o ph os phot h re on ine ) c a n oc c u r

d ur in g tan d em mas s s p ec t r o met r y, t he la tt er d u r in g s amp le pr e pa ra t io n a nd M S a na ly sis .

3

N eu tr al lo s s o f HP O c an a ls o o c cu r d u r in g p o s t‐s o u rc e de c a y of pe p ti de s c on t ain ing

p ho sp h o ser in e an d p ho s p h o t h r eon in e r es i d ue s .

[

27]

identiﬁcation of histidine phosphorylations

567

Specificity for Phosphohistidines

T o de t e r mi ne w he th e r o r n ot t he p r ot o co l w as s p eci ﬁ c fo r p h o s p h o p ep -

t id e s c ont a in in g ph o s ph o r yl at ed Hi s r es i du es , th e Cu ( I I ) ‐I MA C p r o ced u r e

wa s a pp l i e d to a t r yp ti c d ig es t o f b ov in e ꢀ‐ca se in . T h is p r o tei n yi el d s tw o

t ry p ti c pe p ti de s t ha t a r e p ho s p h o r yl at ed at on e an d fo u r S er r es i d u es ,

r e s pe c ti v e l y , a n d w hi ch p r o d uc e io n s o f m/ z 206 2 an d 311 2 wh en an al yz ed

b y po s i ti v e i on M ALD I ‐TO F M S ( C ha lme r s et al . , 200 0; Po s ew it z an d

Te mp st , 1 9 9 9 ) . Ne it h er o f t he s e p ep t id es w as o b s er ved i n Cu ( I I ) ‐I MA C

e x tr a c ts , po s s i bl y b ec au s e t he s t r en gt h o f t he in ter act i o n b etw een p h o s p h o -

2þ

s e r i ne a n d th e i m mo b ili zed C u io n s p r ev ent ed el u t i o n i n 1% N H O H

4

(

N u¨ hs e e t a l., 2 0 0 3 ) . E xt r act io n o f t he ꢀ‐ca s ei n di ge s t u s i ng a G a( I I I ) ‐I MA C

p r o to co l op ti m i z ed f or t he r ec ov er y o f p ep t ide s co n tai n i n g p h o s ph o es t er

r e s i due s ( Po s e wit z an d Te mp s t , 19 99 ) s el e ct iv ely r eco ve r ed th e s i n gly p h o s -

p h or y l a te d pe p ti de o f m/ z 20 62 , as co n ﬁ r med b y p o s it i ve i o n MA L D I ‐T OF

a nd PS D a na l y s i s . Ho we ve r , t he Ga ( I I I ) ‐I M AC p r o to co l d i d n o t r eco ve r th e

H is ‐ph os ph or y l a t ed p e p ti d es f r om t he p ho s p ho r yl at ed H Pr d i ges t, wh i ch

s u gg e s ts th a t i t ma y b e p os s i b le t o u s e Ga ( I I I ) ‐I MA C an d Cu ( I I )‐I MA C

s e qu e nt ia l l y to e x t r a ct p ep t i d es mo d i ﬁ ed w it h p h o s p h o es ter an d p h o s p h o -

h is ti d in e r e s i due s f r om mi xed p r o te in d ig es t s . A lt er n a ti v el y, s te pw is e el u -

t ion of ph os ph ohi s t id i ne s an d p ho s p h o es t er s f r o m Cu ( I I ) ‐I MA C co l u mn s

m ay be po s s ib l e u s in g b as i c s o lu ti o ns o f in cr e as i n g co n cen t r at io n , as s u m in g

t h a t bo th ty p e s of p ho s p h op e pt i d es ar e in d ee d r et ain e d.

Differential Hydrolysis of Phosphohistidines

Hi s ti d i ne i s un iq u e am on g am in o ac id s in t hat i t can b e p h o s ph o r yl ate d

a t tw o di ff e r e nt p os i ti o ns , n ame l y, at at o ms N Á1 an d N e2 o f th e i mi d azo l e

r in g . Fo r fr e e hi s t id i ne , p ho s p ho r y la t io n at t he N Á1 p o s it io n i s m uc h l es s

s ta b le th a n th a t at t he Ne2 p os i t io n d ue t o t he in te r act io n o f th e N Á1 p h o s -

p h a te g r ou p wi th t he p os i ti ve ly ch a r ge d am i no gr o u p ( H ul tq u i s t et al . ,

1

9 6 6 ) . I n pe p ti de s an d p r ot e in s , t he am in o gr o u p o f h i s ti di n e i s i n vo l ved

i n pe p ti de bo nd in g, a n d t he s t ab ili ty o f t he p ho s p h ate l in k ag e m ay d i ffer

co ns i de r a b l y fr o m t ha t o f f r ee h is t id in e. M o re o ve r , th e l o cal s tr u ct ur a l an d

e l e ct r os ta t ic e nv i r o n men t o f t he p r o t ei n ma y h ave s i gn iﬁ can t i n ﬂu e nc e

o n th e c he mi c a l p r o p er t ie s o f h is t id i ne an d may var y i n d i ffer e n t p r o te in

co nt e x ts ( An de r s o n et al ., 19 93 ; W ay go o d et al. , 198 5 ) . Fo r n ati ve H Pr

i n E. c o l i , th e r at e s o f p ho s p ho h y d r ol ys i s f or N Á1 ‐p h o s ph o h i s ti di n e ar e

t h r e e ti me s g r e a te r t ha n t ho s e f or f r ee N Á1 ‐ph o s p h o hi s i ti d i ne (H ul tq u i s t

et a l . , 1 9 6 6 ) , i nd ic at in g t ha t t he p r o t ei n s er v es to d es ta b i l iz e th e l in k ag e,

p e r ha p s to e ns u r e ef ﬁ ci en t p ho s p ho t r an s f er . Th e p H ‐d ep e n d en ce o f p h o s -

p h oh y d r ol y s i s fo r p ho s p ho r y la t ed HPr is al s o q u it e d i s ti nc ti ve , exh i b i ti n g

5

68

genome‐wide analyses of two‐component systems

[27]

a b e l l ‐s h a pe d c ur ve w it h great es t in st ab ilit y of the p hos pho am idate bo nd

betwe en pH 5 a nd 8. Th e p ho sph op ept ide generated b y V 8 di gesti on o f

ph os phory la te d H Pr h as ph os pho hydr olys is r ates an o rder of m agni tude

l ower tha n those o f t he in tact pr ot ein, s how ing greates t s tabi li ty at neutral

t o bas ic pH (Hul tqu is t et al., 1 96 6) . Th is allo ws f or the r etention of p hos phate

du ri ng bas e e lution of t he chelating pipette t ip s.

Summary and Conclusions

Se l e c ti v e e x tr act io n o f Hi s ‐p ho s p h or y la te d p ep ti d es fr o m H Pr p r o tei n

2

þ

d ig e s ts c a n be a ch ie ve d b y im mob i li ze d C u ‐i o n af ﬁn i ty ch r o ma to gr ap h y

u si n g di s p os a bl e me ta l ‐ch e la t in g p ip et t e t ip s . O n ‐ti p aci d i ﬁc ati o n o f th e

s a mp le by r e s i dua l ac et ic ac id ap p ar e nt ly in h i bi ts b i nd i n g o f u n p ho s p h o r y-

l a te d hi s ti d i ne r e s i d ue s d ur i n g C u( I I )‐I M A C , wh i l e r eco ve r y o f th e i n tact

p h os p ho pe p ti de v ia b as e el u ti on is co n ﬁr m ed us i n g n ega ti ve ‐i o n MA L D I ‐

TO F ma s s s pe c t r o met r y. S ub s e qu e n t PS D a n al ys is o f th e p r o to n at ed

m o l ec u l a r i on s h o w s ch ar a c t er is t ic lo s s o f t h e H PO m oi et y p r es en t i n

3

H is ‐ph os p ho r y l a t ed p ep t i d es . Po s s i b le r eﬁ n em en ts to th i s m eth o d i n clu d e

t h e us e of ‘ ‘ co ole r ’’ an d /o r le s s ac id ic ma tr i x co m po u n d s , s u ch as 2, 4,

6

‐tr i hy d r ox y a ce t op h en o n e ( THA P) co mb in e d wi th am mo n i u m ci tr at e

(

Wo l s c hi n e t a l . , 20 05 ) o r 2, 5 ‐d ih yd r o x yb e n z oi c aci d ( D HB ) co m bi n ed

wi th ph os ph or i c ac id (K je ll s t r om an d Je n se n , 200 4 ) , to en h an ce d ete ct i o n

a nd s tr u ctu r a l a na ly s is o f Hi s ‐p ho s p h or y la t ed p ep ti d es b y MA L D I‐T OF

M S, or by M AL D I ‐M S /M S u s in g Q ‐TO F o r TO F ‐T OF i n s t r u me n ta ti o n .

Al th o ug h di s po s ab le p ip et t e t ip s ar e id e al fo r r ap i d I MA C, th e ch ar -

a cte r i s ti c s of th e co lu mn p ac ki ng t en d t o va r y f ro m o n e ti p to an o th er . A s a

r e su lt , th e r e i s s om et im es a la g in s o lv en t u pt a k e wh en as p i r ati n g s o l ut i o ns ,

wh i c h ma y r e s ul t in ai r b ei ng ac ci d en t all y d r aw n i n to th e ti p . I f s o l ut i o ns ar e

a s p i r a te d a nd di s p en s ed w it h ca r e, h ow e v er , th e p ack i n g m ater i al wi l l

r e m a i n pr o pe r l y co n di t io n ed , an d co n s is t en t r es u l ts s h o u ld b e o b tai n ed .

A n a l te r n at i v e a pp r o a ch is t o u s e o pe n t ub u la r ( O T) co l u m n s , i n wh i ch th e

i nn e r s ur f ac e of a gl as s t ub e is u ni f or ml y mo d i ﬁ ed b y ch em ic al at tach m e n t

o f a me ta l c he l a ti n g gr o u p ( Li u et al . , 20 04 ) . OT co l u m n s can b e u s ed i n th e

s a me wa y a s di s po s ab l e p ip et t e t ip s , t he h ig h d en s i ty an d u n if o r m it y o f th e

O T ‐I MA C me d ium p r ov id in g en h an ce d s p eci ﬁ ci ty an d r ep r o d u ci b i li ty fo r

p h os p ho pe p ti de en r i c h men t .

W hi l e pr e l i m in a r y r es u lt s ar e en c ou r ag i n g, th er e i s s ti l l m uc h wo r k to

b e do ne be fo r e r o u t in e, p r ot e om ew id e id e nt iﬁ c ati o n o f H is ‐p h o s p h o r yla te d

p ro te i ns c a n be ac h ie ve d . Al th o u gh f r act i o nat io n o f co m pl ex s am p l es

wo ul d c e rt a i nl y b e ad va n ta ge o us , ea ch ad d iti o n al s te p i n th e an al yt i cal

p ro ce s s i nc r e a s e s t he ch an ce o f d eg r ad i n g p ho s p h o hi s t i d in e r es i d u es , u n le s s

m il d a nd ( pr e fe r a b l y) b a s ic co n d it io n s ca n b e mai n tai n ed th r o u gh o u t . T h is

[

27]

identiﬁcation of histidine phosphorylations

569

p r e cl u de s te c hn i q u es s u ch as S CX , S PE, an d d er i vat i zat io n p r o ced u r e s

f o r e nh a nc i ng r e c ov er y , d e t ect io n , an d /o r r el at i ve q u an ti ﬁ cat io n o f p h o s -

p ho pe p ti de s . Ho w ev er , d if f er e n ti al ce n tr if u ga t io n , tw o ‐p h as e p a r ti ti o n in g ,

a nd / or M OA C m ay p r o ve s u it ab l e f or f r act io na ti on an d co ‐en r i c h me n t o f

H is ‐ph os ph or y l a t ed an d o th e r p ho s p ho r y la t ed p r o tei n s . G el el ec tr o p h o -

r e s i s c omb i ne d w it h p ho s p ha t e ‐s p eci ﬁ c ﬂ uo r e s cen t s ta in i n g , o r We s t er n

b lo tti n g fo r Hi s ‐ an d Ty r ‐p ho s p h o r yl at ed p r ot ei n s , co u l d th en b e u s ed to

r e s ol v e a nd de t e c t p ho s p ho p r o te i n s in t he en ri ch ed fr a c ti o n s . S u b s eq u en t

i de n ti ﬁc a ti on of H is ‐p ho s p ho r y la t ed p r ot e in s , an d th ei r co m po n en t p h o s -

p h oh is t id i ne s , by ma s s s p ect r o me tr y w ou l d b e fa cil i ta te d b y Cu ( I I )‐I MA C

e nr i c hme nt of Hi s‐p ho s p ho r y la t ed p ep t i d es f r o m i n‐gel d i ges ts . H ow ever ,

t h i s a pp r oa c h r e q ui r es t he d ev el op m en t o f r ap i d i n‐gel d i ges ti o n an d

p ep ti de e x tr a c ti o n p r oc ed u r es t ha t mi ni miz e h yd r o l ys is o f p h o s p h o am id at e

b o nd s . Al te r n a ti v el y, ‘‘ to p ‐d ow n ’ ’ an al ys i s b y EC D an d FT ‐I CR MS m ay

p r o v e e ff e c ti v e i n id e nt if y in g an d ma pp i n g la b ile p h o s p h o hi s t i d in e r es i d u es

i n Hi s ‐ph os p ho r y la te d p r ot e in s , p r ov id e d t ha t i n di vi d u al p r o tei n s can b e

i so la t e d or r e co v er ed in t a ct f r om p ol ya c r yl ami d e gel s ( b y el ect r o elu t i o n ,

f o r e x a mp l e ) . T he la t te r w ou ld al s o en a bl e t he u s e o f r ap i d m icr o co l u mn

d ig e st io n pr o ce d ur e s ( S ly s z an d S ch r ie mer , 20 05 ) fo r ‘ ‘b o tt o m ‐u p ’’ an al ys i s

o f Hi s ‐ph os p ho r yl at ed p r ot e i n s .

Un ti l no w, the id e nt iﬁ ca t io n o f n ov el h is t i di n e k i na s e s h as b een p e r -

f o rme d l a r g el y on t he b as i s o f s eq u en ce s im ila r iti es am o n g m em b er s o f th i s

e n z y me c la s s . M il d p r o te in f r ac t io n at io n , ge l s e p ar at i o n, an d af ﬁn i ty p u r i ﬁ -

ca ti o n pr o c e du re s , co mb i n ed w it h h ig h ‐r es o lu t i o n MS an d d ata‐d e p en d en t

n

M S te c hn i qu e s , o ff e r an al t er n at iv e ap p r oa ch fo r i d en t i fyi n g th es e an d

o t he r Hi s ‐ph os p h or y la te d p r ot e in s t hr o u gh d ir ect an al ys i s o f h i s ti di n e p h o s -

p ho r y la ti on s i n p r ot e ol yt i c p ep t id es an d /o r th ei r p ar en t p r o te in s . S u c h

t e ch ni q ue s ha v e t he p ot e nt i al t o in cr e as e s ig ni ﬁ can t l y o u r u n d e r s tan d i n g

o f th e r ol e pl a y e d b y h is t id in e p ho s p ho r y la t io n i n tw o ‐co m po n en t s i gn a l in g

s y s te ms . I n c on cl us i on , I s h ou ld li k e t o t ha n k D r . S co tt N ap p er at th e

V a cc i ne a n d I nf ec ti o us Di se as e Or g an iza t io n ( V I D O) an d D r s. L ia n gl u

W a n a nd Um a K. Ar y a l at t he N R C Pla n t Bi o tec h n o lo gy I n s ti tu t e i n

S as k a to on fo r th ei r as s i s ta n ce in p r ep ar i n g t hi s ar t icl e, wh i ch i s co n tr i b u ti o n

4

8 4 2 1 fr o m th e N at i on al R es e ar ch C ou n ci l o f C an ad a .

References

Ae b er s old , R. , an d M an n , M. ( 20 0 3) . Ma s s s p ec t r o metry ‐ba s ed pr ot e omic s. N a tur e 422,

198–207.

An d er so n , B. , Weig el, N ., K u n di g, W. , a nd R o s ema n, S . ( 19 7 1 ). Su g ar t ra ns po rt . II I P ur iﬁc a -

tio n an d p r o pe rt ie s o f a p h o s p h o car r ie r p r o te in ( HP r) of t he ph os ph oe n olpy ru va t e‐

d ep en d en t p h o sp h o tr an s fe r as e s ys t e m o f E s ch e r ic h ia c oli. J . Bio l. Ch e m. 246, 7 0 23 – 7 03 3 .

5

70

genome‐wide analyses of two‐component systems

[27]

An der s o n , J . W. , Bh an o t , P ., Ge o r ge s, F ., K lev it, R . E ., a nd W ay g oo d, E. B. (1 9 9 1 ). In v olv e -

men t o f th e c ar bo xy‐t er min a l r es i d ue in t he a ct ive s it e of t he hi st idin e‐c on t ain ing pr ot e in ,

HP r , o f th e p h o s p h o en o lp yr u v at e: s u ga r p h o s p h o tr an sf e ra s e sy s t em of Es c he ri chia c oli.

Bio c h emi s tr y 30, 96 01 –9 60 7.

An d er s o n , J . W. , Pu lle n, K ., Ge o r ge s , F ., K lev it, R . E ., a nd W ay g oo d, E. B. (1 9 9 3 ). T he

in vo lvement o f th e a rg in ine 1 7 r es i d ue in t he a ct ive s it e of t he hi st idin e‐c on t ain ing pr ot e in ,

HP r , o f th e p h o s p h o en o lp yr u v at e: s u ga r p h o s p h o tr an sf e ra s e sy s t em of Es c he ri chia c oli.

J . Bio l. Ch e m. 268, 1 23 25 –1 23 33.

An d er s s o n , L. , an d Po r a th , J . ( 19 8 6) . I s o la tio n o f p h o s p ho pr ot e ins by im mo biliz e d m et a l

þ

3

Fe ) afﬁ n ity c hr o mat o gr ap h y. A n al. B io ch e m. 154, 2 50 – 25 4.

(

Ar n o ld , F. H. ( 1991) . Me ta l‐a fﬁ n it y s ep a r at io ns : A n ew di me ns ion in pr ot e in pr oc e s sin g.

Bio te ch n o lo gy 9, 15 1– 15 6.

Ban n ai, H. , Tam ada, Y ., Ma r u ya ma , O. , N ak a i, K ., a nd Miy a no, S. (2 0 0 2 ). Ex t e ns ive f e at u re

d etec tio n o f N‐ter min al p r o te in s o r ti n g s ig n als . B io in fo r ma tic s 18, 2 9 8– 3 0 5.

Beau s o lei l, S . A. , J ed r yc h o w ski, M. , S c h wa r tz , D. , E lias , J . E. , V ill e´ n, J . , L i, J . , Co hn , M . A .,

Can tley, L. C. , an d Gy gi, S . P . ( 20 0 4) . La r ge‐s c ale c h ar ac t e riz a tio n of H eL a c e ll nu c le ar

p h o sp h o p r otein s . Pr o c . N atl. A ca d . S c i. U S A 101, 1 21 30– 12 1 35 .

Br o k x, S . J ., Tal b ot , J . , Go e r ge s , F ., a nd Wa yg o o d, E . B . ( 20 00 ) . En z ym e I of t he ph os ph oe n ol-

p yr u vate: sugar p h o s p h o tr an s fe r as e s ys t e m. I n v itr o in t r a ge n ic c om ple m e nta t ion : T he ro le s

o f Ar g126 in p h o s p h o ry l t r an s f er a nd t he C ‐t er min a l d o ma in in di me riz a tio n. Bio c he m istr y

39, 3624–3625.

Chal mer s , M ., Ro s s , A. R . S . , Ols o n , D. , a nd Ga s k ell, S. J . (2 0 0 0 ). Co mp a ring G a( II I)‐

I M AC meth o d s f or s el ec ti ve e xt r ac tio n a nd MALD I‐T OF M S c ha r ac t e riza t ion of

p h o sp h o p eptid es . Pr o c e ed ing s o f th e 4 8 A S MS C on fe r e nc e, L ong Be a c h, CA , J un e

th

12–15.

Co h en , P. ( 2000) . Th e r eg u la tion o f p r o te in f un c t io n b y mu lt isit e ph os ph ory la tio n— A 2 5‐y e ar

u p d ate. Tr e n d s Bi o ch e m. S c i. 25, 5 96 –6 01 .

Co n r ad s , T. P. , I s s aq , H. J . , a nd V ee n s tr a, T. D. ( 20 0 2) . N e w t oo ls f or qu a nt it at iv e ph os pho-

p r o teo me an alys i s. B io ch e m. B io p hy s . R es . C om mu n. 290, 8 8 5– 8 9 0.

Co o p er , H. J ., Ak b arz ad eh , S . , He alt h , J . K ., a nd Z elle r , M. (2 0 0 5 ). D at a‐de p en de n t e le ct r on

c apt ur e d is s o c iatio n F T‐I C R ma s s s p ec t r o met r y f or p ro t eo mi c a na ly se s . J . P ro teo me Re s .

4, 1538–1544.

d e la Fu en te van Ben t em, S . , R o iting er , E ., An r a th e r , D ., Cs a sz a r, E. , a nd H irt , H . (2 0 0 6 ).

Ph o s p h o pr o teo mic s a s a t oo l t o u n r av el p la n t r eg u la to r y m ec h an ism s. P hy sio l. P lan taru m

126, 110–119.

Eman u els s on , O. , Ni els e n , H. , B r un a k , S . , a nd v on He ijn e , G . (2 0 0 0 ). P re d ict ing su bc e llu lar

lo c aliz atio n o f p r o te in s b as e d o n t he ir N ‐t er min a l a mino a c id se q ue nc e s . J . M ol. Bio l. 300,

1005–1016.

Emme tt, M . R. ( 2003 ) . De te r min at ion o f p o s t‐t r an s la tio n al m odi ﬁc a ti ons of pr ot e in s by hi gh‐

s en s itivi ty, h igh‐r es o lu t io n F o u r ier t r an s f or m io n c yc lo t ron re s on an c e m as s sp e c tr om e try .

J . Ch r o matogr . A. 1013, 2 03 –2 13.

F ic ar r o , S . B. , M cClela n d, M. L. , S tu k e n b er g, P . T. , B ur k e , D . J . , R os s, M . M . , Sh a ba now it z , J . ,

Hu n t, D. F. , an d Wh it e, F . M. ( 20 0 2) . P h o s pho p r o te o me a na ly sis by m as s sp e c tr om et ry a nd

its ap p lic atio n to S ac c h ar o myc e s c e r ev is iae. N at. B io tec h nol. 20, 3 0 1– 3 0 5.

Gr ø n b o r g, M ., Kr i s tia n s o n , T. Z ., S te n s b al le, A. , An d er se n, J . S. , O ha ra , O ., M an n, M . ,

J ens en , O. N. , an d P an d e y, A. ( 20 0 2) . A ma s s s p ec t r om e try‐ba s ed pr ot e om ic a pp ro ac h

fo r id en tiﬁc atio n of s er i n e/t hr eo n i ne‐p h o s p h o ry lat ed p r ot e in s by e nr ic hm en t wit h ph os ph o‐

s p eci ﬁ c an tib o d ies: I d en t iﬁ c at ion o f a n o ve l p r o te in , F r ig g , a s a pr ot e in ki na se A su bs t ra t e.

M ol . Ce ll. Pr o te o mic s 1, 5 17 –5 27 .

[

27]

identiﬁcation of histidine phosphorylations

571

Gr uh l er , A. , Ols en , J. V ., Mo h a mme d, S . , Mo r t en s e n , P ., F a er g em a n, N . J . , M an n, M . , a nd

J ens en , O. N. ( 20 05 ) . Qu a nt it ativ e p h o s p h o p rot eo mics a pp lie d t o t he y e as t ph e ro mo ne

s ign alin g p ath way. Mo l. C ell . Pr o te o mics 4, 3 10 –3 27 .

Han s o n , B. J ., S ch u len b e r g, B ., P at to n , W. F ., a nd C ap a ld i, R . A . (2 0 0 1 ). A no v el su bf r ac t io na -

tio n ap p r o ach fo r mit o c ho n d r ia l p r o te in s : A t hr e e‐d im en sio na l m ito c ho nd ria l pr ot e om e

map . Ele ct r o p ho r es is 22, 9 50 –9 59 .

Hes s , J . F. , Bo u r r et, R. B ., a nd S imo n , M. I . ( 19 8 8) . His t id ine ph os ph ory la tio n a nd ph os ph or yl

gr o u p tr an s fer in b ac t er ial c h emo t axis . N atu r e 336, 1 39 –1 4 3.

Ho d ges, T. K. , Leo n ar d, R . T. , B r ac k er , C . E ., a nd K en n a n, T . W . (1 9 7 2 ). P ur iﬁc a t ion of a n

io n ‐s tim ul ated ade n o s in e t r ip h os ph at a s e f r om p la n t ro ot s : A ss oc ia tio n wit h pl as ma

memb r an es . Pr o c . N atl. A ca d . S c i. U S A 69, 3 30 7– 33 11 .

Hu lt qu i st, D. , M oy er , R . W. , a nd B oy e r, P . D. ( 19 6 6) . Th e p re p ar a ti on a nd c ha r ac t e riz a tio n of

1‐p h o sp h o his ti d in e a nd 3‐p h o s p h o h is tid in e . B io ch e mis t ry 5, 3 2 2– 3 3 1.

J ane k, K. , Wen s c hu h , H. , B ien e r t, M. , a nd K r au s e , E . ( 20 0 1 ). P ho sp ho pe pt ide a na ly sis by

p o s itive an d n ega tive io n ma tr ix‐a ss i s te d la s er d es o r p tio n/io niz a t ion m as s sp e c tr om et ry .

Rap id Co mmu n. M as s S p e ct r o m. 15, 1 59 3– 15 99 .

K je lls tr o m, S ., an d J ens e n , O. N . ( 20 0 4) . P h o s pho r ic a cid a s a m at r ix a dd it ive f or M AL DI M S

an alys i s o f p h o sp h o p ept id es a nd p h o s p h o p rot ein s . A n al. Ch e m. 76, 5 1 09 – 5 11 7 .

K lu mp p, S ., an d Kr i egls t ein , J . ( 20 0 2) . P h o s ph o r yla tio n a nd de p ho sp ho ryla t ion of hi st idin e

r es id u es in p r o tein s. E ur . J . B io ch e m. 269, 1 06 7– 10 71 .

L ars s o n , C. , Wid ell , S . , a nd K jell b o m, P . ( 19 8 7) . P r ep a r atio n of hi gh pu rit y pl as ma m em br a ne s.

M eth . En zymo l. 148, 5 58 –5 68 .

L auges en, S ., M es s in es e , E ., He m, S . , P ic h ere au x , C ., Gr a t, S. , R a nje v a, R . , R os sig n ol, M . , a nd

Bo n o , J . J . ( 2006) . P h o s ph o p r ot ein a na ly s is in p la n ts : A pr ot e om ic a pp ro ac h. P hy toc h e mis-

tr y 67, 2208–2214.

L iu, H. , S tu p ak , J ., Z he n g, J . , K elle r , B . O. , B r ix, B . J . , F lie g el, L ., a nd L i, L . (2 0 0 4 ). O pe n

tu b u lar imm o bi liz ed me ta l io n a fﬁ n it y c h r o mat og ra p h y c om bin e d wit h M AL DI M S a nd

M S/ M S fo r id en tiﬁ c at ion o f p r o te in p h o s p h o ry lat io n s ite s . A na l. Ch e m. 76, 4 2 23 – 4 23 2 .

Ma tth ews , H. R. ( 1995 ) . P r o te in k in a s es a nd p h o s p h ata se s t ha t a c t on hi st idin e , ly sin e , or

ar gin in e r es id u es in e uk a r yo t ic p r o te in s : A p o s s ib le re g ul ator of t he m ito g en‐a c tiv a t ed

p r o tein k in as e c asc a d e. Ph a r mac . Th e r . 67, 3 23 –3 50 .

Me ad ow, N. D. , Fo x, D. K ., a nd R o s ema n, S . ( 19 9 0) . Th e ba c t e ria l ph os ph oeno lpy r uv at e :

glyc o s e p h o sp h o t ran s fe r as e s ys t e m. A n n u. R ev . B io ch e m. 59, 4 9 7– 5 4 2.

N ap p er , S ., Br o k x, S . J . , P all y, E ., K in d r ac h u k , J . , De lb a er e , L . T . J . , a nd W ay g oo d, E. B.

(

2001) . S u b st itu tio n o f a sp a r ta t e a nd g lu ta mat e f or a c tiv e c e nt e r hi st idin e s in t he

Es c he r ic hi a c ol i p h o s p h o en o lp yr u v at e: s u ga r p h o s p h o tr an sf e ra se sy s t em m ain t ain ph os ph o-

tr an s fer p o ten tial. J . B io l. C he m. 276, 4 15 88 –4 15 93.

N ap p er , S ., Ki n dr ac hu k , J . , Ols o n , D. J . H. , Amb r o s e , S . J . , D er e niws ky, C. , a nd R os s, A . R . S.

(

2003) . S elec tive e xt r ac tio n a nd c h ar a ct er iz at io n o f a hi st idin e‐ph os ph or yl at e d pe p ti de

u s in g imm o bi liz ed c o p p er ( I I )‐io n a fﬁ n it y c h r o mat og ra p h a nd m at r ix‐a ss ist e d la se r de s or p-

tio n /i o n izat io n‐tim e o f ﬂ ig h t ma s s s p ec t r o met r y. A n al. Ch e m. 75, 1 7 41 – 1 74 7 .

N u¨ h s e, T. S ., S ten s b al le, A. , J en s e n , O. N ., a nd P ec k , S . C. (2 0 0 3 ). L ar g e‐sc a le a na ly sis of

in vivo p h o sp h o r ylat ed me mb r an e p r o te in s b y immo b il iz ed m et a l io n a f ﬁni ty c hr om a tog -

r ap h y an d mas s s p ec t r o metr y. Mo l. C ell . Pr o te o mics 2, 1 2 34 – 1 24 3 .

P ark i n son , J . S ., an d K o fo id , E . C . ( 19 9 2) . C om mu nic at i on m odu le s in ba c t e ria l si gn a ling

p r o tein s . An n u . Re v. Ge n e t. 26, 7 1– 11 2.

P at el, H. R. , an d Gel fa n d , E . W. ( 19 9 6) . DN A‐b in d in g p h os ph op rot e ins in duc e d a f te r T c e ll

ac tivatio n : Effec ts o f c yc lo s p o r in A. C ell . S ig n allin g 8, 2 5 3– 2 6 1.

P ers s o n , A. , an d J er gil, B . ( 19 9 2) . P u r iﬁ c at io n o f p la s ma m em br a ne s by a qu eo us t wo ‐ph a se

afﬁ n ity p ar titio nin g . A n al. B io ch e m. 204, 1 31 –1 36.

5

72

genome‐wide analyses of two‐component systems

[27]

P o s ewitz , M . C. , an d Te mp s t, P . ( 19 9 9) . I mmo b iliz e d g alli um (I II ) a f ﬁni ty c hr om a to g raph y of

p h o sp h o p eptid es . A n al. C he m. 71, 2 88 3– 289 2.

P os tm a, P. W. , Len gele r , J . W. , a nd J ac o b s o n , G. R . ( 19 9 3 ). P ho sp ho en olp yr uv a t e: c a rboh y -

d r ate p h o sp h o t ran s fe r as e s ys t e ms o f b ac t er ia . Mic r o b io l. Re v . 57, 5 4 3– 5 9 4.

R os s , A. R. S ., Lee, P . J . , S mit h , D. L. , La n gr id g e, J . I . , W he t to n, A . D ., a nd G as ke ll, S. J .

(

2002) . I d en tiﬁ c at ion o f p r o te in s f r om t wo‐d ime n s io nal po ly a cr y la mid e g e ls us ing a no v el

ac id‐lab il e s u r fac ta n t. Pr o te o mic s 2, 9 28 –9 36.

R us h , J ., M or i tz, A. , Le e, K . A. , Gu o , A. , Go s s , V . L. , Sp e k, E. J . , Zh an g , H ., Zh a, X . M . ,

Po l ak iewic z, R. D ., a nd C om b, M. J . ( 20 0 5) . I mmu n oa fﬁ nit y pr oﬁ ling of t y ros in e ph os ph or-

ylat io n in c anc er c ell s . N at. B io tec h n o l. 23, 9 4– 10 1.

S c hin d ler , J ., Lewan d r ow s ki , U. , S ic k man n , A. , F r iau f , E. , a nd N ot h wa ng, H . G . (2 0 0 6 ).

Pr o teo mic an alys is o f b r ai n p la s ma me mb r an es is o la te d by a f ﬁni ty t wo‐ph a se pa r ti tio nin g .

M ol . Ce ll. Pr o te o mic s 5, 3 90 –4 00 .

S c hwep p e, R. E. , Ha yd o n , C . E ., Le wis , T. S . , R es in g , K. A ., a nd A hn, N . G . (2 0 0 3 ). T he

c har ac ter iz atio n of p r o te in p o s t ‐t r an s la tio n a l mo d iﬁ c at ion s by m as s sp e c tr om et ry . A c c .

Ch e m. Re s . 36, 453 –4 61.

S lys z , G. W. , an d S ch r ieme r , D. C . ( 20 0 5) . B len d in g p r o te in se p ar a t ion a nd pe p ti de a na ly sis

th r o u gh r eal‐tim e p r o te o lyt ic d ig es t io n . A n al. C he m. 77, 1 5 72 – 1 57 9 .

S teen , H. , Fe rn an d ez, M. , Gh a ff ar i, S . , P an d e y, A. , a nd Ma nn , M . (2 0 0 3 ). P ho sp ho ty r os ine

map p in g in Bc r /Ab l o n c o pr ot ein u s in g p h o s p h o tyr o s in e‐sp e c iﬁc im mo niu m io n sc a n nin g.

M ol . Ce ll. Pr o te o mic s 2, 1 38 –1 45 .

S to ck , A. , Ko s h lan d , D. E ., J r . , a nd S to c k , J . ( 19 8 5) . Ho m olog ie s be t we e n t he Sa lm on e lla

typ h imu r iu m Ch e Y p r o te in a nd p r o te in s in v o lve d in th e re g ul at io n of c he m ot a xi s, m em -

b r an e p r o tein s yn th es i s , a nd s p o r u lat ion . Pr o c . N atl. A ca d. Sc i. U SA 82, 7 9 89 – 7 99 3 .

S to ck , J ., Ni n fa, A. , a nd S to c k , A. M. ( 19 8 9) . P r o te in p ho sp ho ry lat ion a nd re g ul at io n of

ad ap tive r es p o n s es in b ac t er ia. Mic r o bio l. R ev . 53, 4 50 – 49 0.

T it gemeyer , F. ( 1993) . S ig n al t r an s d u c tion in c h emo t ax is m ed ia te d by t he ba c t e rial ph os ph o-

tr an s fer as e s ys tem . J . C ell . B io ch e m. 51, 6 9– 74 .

V an Pu tte, R. D., an d Pat te rs on , C. O. ( 200 3). Mic ro alga l p lasma me mbranes puriﬁed by a queous

two‐p hase partitioning . Tran sa ctio ns o f the Illino is S ta te Aca de my of Science s 96, 7 1– 86 .

V yet r og on , K. , Teb b ji, F ., Ols o n , D. J . H. , R o s s , A. R . S. , a nd M at t on , D .P (2 0 0 6 ).

A c om pa rative pr o t eo me a nd p h o s p h o pr o te o me a n al ys is of di ff e re n tia lly re g ul at e d

p r o tein s d u r in g fe r tiliz a tion in t he s el f in c o mp at ib le sp e c ies So la nu m c ha c oe nse Bit t .

Pr o te o mics 7, 232– 24 7.

Waygo o d , E. B. , Er ik s on , E . E ., E l‐K ab b a n i, O. A. L. , a nd D elb a er e , L . T . J . (1 9 8 5 ). Ch ar a c -

ter iz atio n o f p h o s p h o ry lat ed h is t id in e‐c o n ta in in g p r o te in (H P r) of t he ba c t e rial ph os pho-

en o lp yr u vate/s u ga r p h os p h o tr a n s fe ra s e s ys t e m. B io ch e m istr y 24, 6 9 38 – 6 94 5 .

Wo ls c h in, F. , Wien k o op , S . , a nd We ck w er t h , W. ( 20 0 5 ). En ric h me nt of ph os ph ory la te d

p r o tein s an d p ep ti d es f r om c o mp lex mix tu r e s u s ing m et a l ox id e /hy dr ox ide a f ﬁni ty

c hr o matogr ap h y ( MOAC ) . Pr o te o mic s 5, 4 38 9– 43 97 .

Wo lte rs , D. A. , Was hb u rn , M. P ., a nd Y at es , J . R ., I I I ( 20 0 1 ). A n a ut om a t ed m ult idime ns ion al

p r o tein id en tiﬁ cat io n t ec h n o log y f or s h o tg u n p r o te o mics. A na l. Ch e m. 73, 5 6 83 – 5 69 0 .

Wu en s ch ell , G. E. , N ar a n jo , E ., a nd Ar n o ld , F . H. ( 19 9 0) . A que o us t wo‐ph a se m et a l a f ﬁni ty

extr ac tio n o f h em e p r o te in s . B io p r oc e s s E ng in e e r in g 5, 1 9 9– 2 0 2.

Y at es , J . R. , I I I ( 1998) . Ma s s s p ec t r o met r y a nd t he a ge o f th e pr ot e om e . J . M as s Sp e c tro m. 33,

1–19.

Article Doi

Biomass toward fine chemical products: Oxidation of α-pinene over sieves nanostructured modified with vanadium

DOI: 10.1016/j.molcata.2015.04.009

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.molcata.2015.04.009

Full text of DOI:10.1016/j.molcata.2015.04.009

Products guided by the article

R&D Labs maybe for 13040-03-4

Relevant to this article

Hot Product