Af

AD

R

bttdnagictafmsaA

p

pppdppatpcttc

3

1

Analytical Biochemistry 266, 198–204 (1999)Article ID abio.1998.2955, available online at http://www.idealibrary.com on

Two-Dimensional Peptide Gel Electrophoresis Systemor Phosphopeptide Mapping and Amino Acid Sequencing

ndrea Gatti1 and Jolinda A. Traughepartment of Biochemistry, University of California, Riverside, California 92521

eceived July 10, 1998

ppp

pdhpr

tvCaavsmasgmfT

gmrfigitpcsr

A novel two-dimensional electrophoresis system toe carried out on polyacrylamide gels under nondena-uring conditions was developed to efficiently frac-ionate the peptides resulting from endoproteinaseigestion of 32P-labeled proteins. In particular, nonde-aturing gel isoelectric focusing was combined withlkaline 40% polyacrylamide gel electrophoresis toenerate phosphopeptide maps with high reproduc-bility, thus allowing both protein fingerprinting andomparative analysis of different samples. The poten-ial application of this method for subsequent aminocid sequencing of the isolated phosphopeptides wasurther demonstrated by successful manual and auto-ated Edman sequencing. Taken together these data

how that such a simple and precise approach is suit-ble for both analytical and preparative aims. © 1999

cademic Press

Key Words: two-dimensional phosphopeptide map-ing; isoelectric focusing; alkaline polyacrylamide gel.

One of the most useful applications of protein finger-rinting consists of the comparative analysis of phos-hopeptide maps resulting from proteolyzed phospho-roteins. A number of distinct techniques have beenescribed to provide a means for fractionating phos-hopeptides and they are all based on the cleavage ofroteins with defined endoproteinases or chemicalgents followed by a one- or two-dimensional separa-ion of the resulting peptides. While the resolution ofeptide fractionation achieved by one-dimensional pro-edures is often insufficient to analyze complex mix-ures of peptides, the conventional two-dimensionalechniques suffer from the difficulty of efficiently re-overing uncontaminated peptides. To overcome these

1

To whom correspondence should be addressed. Fax: 909-787-590. E-mail: andrea@pop.ucr.edu. h

98

roblems we have developed a phosphopeptide map-ing system that is suitable for both analytical andreparative purposes.Here we describe a novel combination of two distinct

hosphopeptide mapping gel techniques, namely non-enaturing gel isoelectric focusing (1) and alkalineigh reticulation PAGE (2), to fractionate the phos-hopeptides along two dimensions and generate high-esolution phosphoprotein fingerprints.To develop our experimental strategy, we employed

he serine/threonine protein kinase g-PAK2 (p21-acti-ated kinase, also known as PAK2 or PAK I), afterdc42-stimulated autophosphorylation (3, 4). g-PAK isn isoform of the PAK family, whose protein kinasectivity is known to be regulated by binding to acti-ated members of the Rho family of small GTPasesuch as Cdc42 (5). The choice of using g-PAK as aodel phosphoprotein is due to the evidence that PAKs

re characterized by multisite phosphorylation, witheveral autophosphorylated sites in the N-terminal re-ion (6). For comparative purposes, a N-terminal frag-ent of g-PAK (g-PAK1–96) was also used as a substrate

or g-PAK, as described elsewhere (Huang, Z., andraugh, J. A., unpublished data).After in vitro phosphorylation, both g-PAK and

-PAK1–96 were subjected to cleavage with two com-only used endoproteinases (trypsin and Lys-C). The

esulting phosphopeptide maps allowed comparativengerprinting of the N-terminal region and full-length-PAK, revealing the high resolution and reproducibil-ty of this mapping system. Further, the feasibility ofhis approach for preparative as well as analytical pur-oses was shown by employing a simple cleanup pro-edure to isolate the individual phosphopeptides in auitable form for manual and automated Edman deg-adation techniques. This strategy enabled us to iden-

2

Abbreviations used: PAK, p21-activated protein kinase; HPLC,igh-performance liquid chromatography.

0003-2697/99 $30.00Copyright © 1999 by Academic Press

All rights of reproduction in any form reserved.

toeT

E

M

dPLhTp(wttqgaH

P

mcwt(tM[a3Cro

T

ttipowdvam

E

wLec1bndt

T

jsaN(3aai1a2mt4wlcgrls(kcbS1wgmv

ttctPg

199TWO-DIMENSIONAL PEPTIDE GEL ELECTROPHORESIS

ify a number of autophosphorylation sites in g-PAK,ne of which is described in detail here and the otherslsewhere (Gatti, A., Huang, Z., Tuazon, P. T., andraugh, J. A., submitted for publication).

XPERIMENTAL PROCEDURES

aterials

All chemicals were from Sigma, if not otherwise in-icated. Ampholines (pH 3.5–10 and 3.5–5) were fromharmacia; Zwittergent 3-16 was from Calbiochem;ys-C endoproteinase was from Boehringer Mann-eim; nitrocellulose was from Schleicher & Schuell.he slab gel unit employed for the second-dimensionaleptide PAGE was from Hoefer Scientific InstrumentsModel SE 400). Cellophane membrane backing sheetsere from Bio-Rad. Arylamine disks (Sequelon AA

ype) were from PerSeptive Biosystems and C18 car-ridges (Sep-Pak, 50 mg) were from Waters. The se-uencer (Procise 492) was from Applied Biosystems.-PAK1–96 was cloned and expressed in Escherichia colind the purified sample was generously provided by Z.uang.

rotein Kinase Assay

Autophosphorylation of recombinant g-PAK (0.5–2g), purified from baculovirus-infected insect cells, wasarried out in the presence of Cdc42 previously loadedith GTPgS, as described elsewhere (4). Phosphoryla-

ion of recombinant g-PAK1–96 (0.5–2 mg) by g-PAK0.05–0.2 mg) was carried out in a 20-ml reaction mix-ure containing 20 mM Tris–HCl, pH 7.4, 10 mMgCl2, 30 mM b-mercaptoethanol, and 0.2 mM

g-32P]ATP (1000 cpm/pmol). The phosphorylation re-ctions were terminated after 30 min incubation at0°C by addition of 100 ml of 9.5 M urea, 5% (w/v)haps, 5% (v/v) b-mercaptoethanol, 0.1 mg/ml methyled, plus pH 3.5–10 ampholines to a final concentrationf 2% (v/v).

wo-Dimensional Protein Gel Electrophoresis

Samples were subjected to a two-dimensional pro-ein PAGE, as previously described (7). Briefly, isoelec-ric focusing in tube gels (7.5 cm in length and 0.2 cmn diameter) was followed by SDS–PAGE in a 10%olyacrylamide gel. Proteins were electrotransferrednto nitrocellulose (8) and the membrane was stainedith 0.5% (w/v) ponceau in 1% acetic acid prior toetection via autoradiography. Cerenkov counting re-ealed that greater than 75% of the counts loaded onto

gel were typically recovered on the nitrocellulose

embrane. a

ndoproteinase Cleavage

The relevant 32P-labeled proteins on nitrocelluloseere excised and digested either with trypsin or withys-C endoproteinase, essentially as described by Luit al. (9). Briefly, digestion of 2–10 mg of protein wasarried out at 30°C for 15–24 h in a final volume of00–200 ml of freshly prepared 50 mM ammoniumicarbonate, 1% (w/v) Zwittergent 3-16, plus 20–50g/ml of the chosen endoproteinase. Under these con-itions, greater than 90% of the Cerenkov counts wereypically released from the nitrocellulose membrane.

wo-Dimensional Peptide Gel Electrophoresis

Aliquots (20–50 ml) of the digested protein were sub-ected to nondenaturing gel isoelectric focusing. The gelolution consisted of 7.5% (w/v) polyacrylamide (usingstock solution with a 30:1.8 ratio of acrylamide and,N9-methylene-bisacrylamide, respectively) and 7.5%

v/v) ampholines (using a 7:3 ratio of pH 3.5–5 and.5–10, respectively). Prior to gel casting, 0.043% (w/v)mmonium persulfate and 0.1% (v/v) TEMED weredded to achieve polymerization. Gel tubes were 12 cmn length and 0.2 cm in diameter. Samples were diluted:1 with 20% sucrose and 4% (v/v) of the above mix ofmpholines, loaded onto gel tubes, and overlayed with0 ml of 5% sucrose, 1% (v/v) ampholines and 0.1 mg/mlethyl red as tracking dye. First-dimensional isoelec-

ric focusing was carried out at room temperature for–5 h at 1 mA/300 V maximum setting and terminatedhen the tracking dye had migrated half of the gel

ength. After removal from the glass tubes, the gelylinder was positioned on top of a 40% alkaline slabel (15 cm wide and 0.75 mm thick), consisting of aesolving gel (12 cm long) and of a stacking gel (3 cmong). Gel solutions and reservoir buffer were as de-cribed by West and colleagues (2), except that 24%v/v) glycerol replaced 6 M urea in the stacking gel. Toeep the gel cylinder in place, 1 ml of reservoir bufferontaining 1% (w/v) agarose was poured in the spaceetween the electrofocused gel and the top of slab gel.econd-dimensional electrophoresis was carried out for2–15 h at 5 mA/gel constant current and terminatedhen the tracking dye had migrated two-thirds of theel length. The gel was then coated with a cellophaneembrane-backing sheet and dried for 15 min under

acuum with heat, prior to autoradiography.For comparative purposes, the tryptic digest of au-

ophosphorylated g-PAK was also subjected to a dena-uring two-dimensional SDS–PAGE system (10),onsisting of isoelectrofocusing in urea/detergent-con-aining gel (7) followed by second-dimensional 16%AGE in Tris–Tricine buffer (11). In such a case, theel was dried under vacuum with heat for 1 h prior to

utoradiography.

E

vbrssnc0w0dtctwt

M

ma(v5cttuifydflrt4td

R

gslndtHfplt

te

pfcaarrol

reaaPwp

g3sbiaopc

t

Figri

200 GATTI AND TRAUGH

xtraction and Cleanup of Selected Phosphopeptides

The radioactive tryptic phosphopeptides were indi-idually excised from the 40% alkaline gel and incu-ated in 2 ml of double-distilled water overnight atoom temperature. The released phosphopeptides wereubjected to a cleanup procedure essentially as de-cribed by Sullivan and Wong (12). Briefly, the super-atant was loaded onto a commercially available C18artridge (50 mg), which was then washed with 5 ml of.1% trifluoroacetic acid, and the radioactive peptideas eluted with 1 ml of 66% acetonitrile containing.1% trifluoroacetic acid. The final eluate was taken toryness in a speed-vac prior to Cerenkov counting ofhe radioactivity. Comparison of the counts in the ex-ised gel slice and the dried eluate from the C18 car-ridge revealed that the recovery of the phosphopeptideas typically close to 90%, with less than 10% varia-

ion between experiments.

anual and Automated Sequencing

The lyophilized phosphopeptide was dissolved in 20l of 30% acetonitrile containing 0.1% trifluoroaceticcid and immobilized on a disk of arylamine membraneSequelon AA type), as described by the supplier. Co-alent linkage was accomplished by adding to the diskml of a solution of ethyl-3-(3-dimethylaminopropyl)

arbodiimide in 0.1 M Mes (pH 5.0). The surface ofhe Sequelon AA membrane reacts with both the C-erminal and side-chain carboxyl groups of peptidespon incubation with water-soluble carbodiimide. Typ-

cally 75% of the radioactivity loaded onto the disk wasound to covalently bind to it, as determined by anal-sis of Cerenkov counts after sequential washing of theisk in double-distilled water, methanol and 100% tri-uoroacetic acid. After being washed, the disk wasoutinely cut in two parts: approximately three-quar-ers of the disk was placed in the reaction vessel of a92 Procise Sequencer from Applied Biosystems, whilehe remaining quarter was subjected to manual Edmanegradation as described by Sullivan and Wong (12).

ESULTS

Following phosphorylation, 32P-labeled g-PAK and-PAK1–96 were isolated via conventional two-dimen-ional electrophoresis and transferred onto nitrocellu-ose membranes. Recombinant g-PAK exhibited aumber of differentially migrating forms upon two-imensional PAGE and this profile changes after au-ophosphorylation, as described elsewhere (Gatti, A.,uang, Z., Tuazon, P. T., and Traugh, J. A., submitted

or publication). For the purpose of this study, theositions of radiolabeled g-PAK and g-PAK1–96 wereocated by ponceau staining and autoradiography of

he blot membranes (data not shown). The phosphopro- g

eins were excised from the nitrocellulose and digestedither with trypsin or with Lys-C endoproteinase.In initial experiments, aliquots of the digested sam-

les were subjected to a single-dimensional isoelectricocusing carried out in tube gels under nondenaturingonditions. Phosphopeptides were therefore fraction-ted on the basis of the difference in pI, with the mostcidic peptides migrating farthest. After gel drying,adiolabeled phosphopeptides were visualized by auto-adiography (Fig. 1). As noted by Hardie et al. (1), somef the resolution of the phosphopeptide separation isost upon gel drying.

Having established suitable conditions for the sepa-ation of phosphopeptides by nondenaturing gel iso-lectric focusing, we then introduced a second fraction-tion step by loading the electrofocused gel on top of anlkaline 40% gel, as described under Experimentalrocedures. The resulting two-dimensional peptide gelas subjected to autoradiography to visualize thehosphopeptides.When such a protocol was applied to the proteolyzed

-PAK, the phosphopeptide maps shown in Figs. 2 andwere obtained with the use of trypsin or Lys-C, re-

pectively. The radioactive spots with assigned num-ers/letters were the major 32P-labeled peptides result-ng from cleavage of autophosphorylated g-PAK. Tollow a comparison between the phosphopeptide mapsf full-length g-PAK and the N-terminal fragment,hosphorylated g-PAK1–96 was run separately and inonjunction with autophosphorylated g-PAK.Regardless of the type of endoproteinase in use,

he phosphopeptides generated from digestion of

IG. 1. Separation of 32P-labeled phosphopeptides by nondenatur-ng gel isoelectric focusing. In vitro phosphorylated samples of-PAK1–96 and g-PAK were digested with Lys-C or trypsin and theesulting phosphopeptides were fractionated by gel isoelectrofocus-ng under nondenaturing conditions. The autoradiogram is shown.

-PAK1–96 migrated similarly to the corresponding

ppm

mgatp

ss(lttTslp

tsifciqpttyot

mptifw

Fttlisfbt

Fde

Ftdw1e

201TWO-DIMENSIONAL PEPTIDE GEL ELECTROPHORESIS

hosphopeptides of g-PAK. Identity between phos-hopeptides was confirmed by comigration experi-ents, wherein aliquots of both digested samples were

IG. 2. Separation of 32P-labeled tryptic phosphopeptides afterwo-dimensional peptide PAGE. The samples were generated byryptic digestion of phosphorylated g-PAK1–96 (A), autophosphory-ated g-PAK (B), and g-PAK1–96 plus g-PAK (C) prior to nondenatur-ng gel isoelectric focusing and alkaline 40% PAGE. (D) shows achematic representation of the major phosphopeptides resultingrom trypsinization of g-PAK1–96 and g-PAK, with those in commoneing shaded. Only the relevant sections of the autoradiograms withhe major phosphopeptides are shown.

IG. 3. Separation of 32P-labeled Lys-C phosphopeptides after two-

fimensional peptide PAGE. Conditions are the same as in Fig. 2,xcept that the employed endoproteinase was Lys-C.

ixed and run on the same two-dimensional peptideel, as shown in Figs. 2C and 3C. Phosphopeptides 1nd 2 resulting from tryptic digestion and phosphopep-ides a, b, and c resulting from the use of Lys-C endo-roteinase were present in both g-PAK1–96 and g-PAK.To compare this procedure with a previously de-

cribed gel system based on a conventional two-dimen-ional electrophoresis under denaturing conditions10), an aliquot of the tryptic digest of autophosphory-ated g-PAK was subjected to a peptide mapping sys-em consisting of a urea/detergent-containing isoelec-rofocusing gel followed by a 16% SDS–PAGE in aris–Tricine buffer system. The resulting two-dimen-ional fractionation of phosphopeptides (Fig. 4) was ofower resolution compared to that achieved with oureptide gel system (shown in Fig. 2B).To utilize the peptides isolated by this phosphopep-

ide mapping system for sequencing purposes, we de-igned a protocol by which the phosphopeptides result-ng from endoproteinase digestion could be extractedrom the second-dimensional gel, subjected to aleanup procedure on Sep-Pak cartridges to removenterfering contaminants, and covalently bound to Se-uelon AA disks. To quantitate the recovery of the twohosphopeptides resulting from in vitro phosphoryla-ion of g-PAK1–96 throughout the phosphopeptide frac-ionation, extraction, and binding to Sequelon AA, theield was determined by comparing the counts/minutef individual phosphopeptides recovered before and af-er each methodological step (Table 1).

As a model phosphopeptide to be subjected to auto-ated and manual sequencing analyses, we focused on

hosphopeptide 2 to determine the major phosphoryla-ion site in g-PAK1–96. Despite some interference in thenitial sequencing cycle due to an excess of glycine, theollowing residues of the examined phosphopeptideere successfully identified by the sequencer, except

IG. 4. Separation of 32P-labeled phosphopeptides by conventionalwo-dimensional electrophoresis carried out under denaturing con-itions. An aliquot of autophosphorylated, tryptic digested g-PAKas subjected to denaturing gel isoelectric focusing followed by a6% SDS–PAGE in Tris/Tricine buffer system, as described by Lopezt al. (10). The autoradiogram is shown.

or the residue at the position corresponding to a can-

docsqatCs(mSt

D

afsessdmpdNsaPrr

tpsssiiatbpS(

pltriT

m ctuo

FpgpEmg

202 GATTI AND TRAUGH

idate phosphorylation site (Fig. 5). Note that the lackf recovery of PTH-serine in a phosphopeptide at aycle known to correspond to serine is indicative oferine phosphorylation (13, 14). Parallel manual se-uencing showed the release of the majority of the 32Pt the third cycle (Fig. 5), thus enabling us to excludehe presence of other phosphorylation sites in theOOH terminus of phosphopeptide 2. With Lys-52 as aite of tryptic proteolysis in the sequence of g-PAK1–96

Fig. 6), the results from automated sequencing andanual Edman degradation unambiguously identifyer-55 as the only phosphorylation site of phosphopep-ide 2 of g-PAK1–96.

TAB

Analysis of Recovery Yields of Try

Phosphopeptide

Amou

Loaded onto2-D peptide gel

Extr2-D p

1 1002 100

a Data are expressed as percentage of the initial radioactivity witarked with an asterisk represent a slight underestimation of the a

nly 15 min of gel drying.

IG. 5. Manual and automated sequencing analysis of phos-hopeptide 2. Sequencing of phosphopeptide 2 from phosphorylated-PAK1–96 was carried out after isolation from the two-dimensionaleptide gel. The amounts of 32P released at each cycle by manualdman degradation and the peptide sequence identified by auto-

pated sequencing are indicated together with the sequence of

-PAK1–96, as deduced from the corresponding cDNA (4).

ISCUSSION

Phosphopeptide mapping has two main applications,nalytical for comparative purposes and preparativeor sequencing analysis. Notably, the procedure de-cribed here can serve both aims by using the samexperimental approach. A recent study (10) has de-cribed the use of a conventional two-dimensional gelystem to fractionate the peptides resulting from en-oproteinase digestion. However, some of the experi-ental conditions described are not ideal for further

rocessing of the phosphopeptides. First, the use ofenaturating agents such as urea and SDS may cause-terminal blockage and interference with automated

equencing, respectively. Second, a standard percent-ge of polyacrylamide in the second-dimensional SDS–AGE (not higher than 20%) does not generate high-esolution maps of peptides as small as those typicallyesulting from exhaustive tryptic digestion (Fig. 4).In contrast, the alkaline high-reticulation PAGE

echnique was originally designed to fractionate smalleptides as a function of their combined charge andize (2). As a matter of fact, such a system has beenuccessfully used to fractionate phosphopeptides in aingle-dimension PAGE (15, 16). Here we report that,f combined with a first-dimensional isoelectric focus-ng step, the alkaline 40% polyacrylamide gel providesn excellent resolution of tryptic phosphopeptides inwo dimensions, thus representing the experimentalasis for successful amino acid sequencing. This wasroven true by the unambiguous identification ofer-55 as the major phosphorylation site in g-PAK1–96

Fig. 5).Indeed, a thorough characterization of g-PAK auto-

hosphorylation in response to activated Cdc42, usingess than 100 pmol of starting protein and employinghe same experimental strategy here described, hasevealed several sites of autophosphorylation, includ-ng Ser-55 (Gatti, A., Huang, Z., Tuazon, P. T., andraugh, J. A., submitted for publication).The fact that on the basis of comparative phos-

1

c Phosphopeptides from g-PAK1–96

f phosphopeptide recovered (%)a

d fromtide gel

Eluted fromC18 cartridges

Bound toSequelon AA

* 57 38* 52 32

espect to the counts recovered at each following step. The numbersal content of radioactivity because the gel slices were counted after

LE

pti

nt o

acteep

6458

h r

hopeptide mapping we could correctly predict the

itstpatT

qitdp

itppdlscpipfdptpspcampd

tptms

eyprod

asserye(btuamcqsmbiS

etpigTcaa

sgpipiacbi

Fqcgp

203TWO-DIMENSIONAL PEPTIDE GEL ELECTROPHORESIS

dentity between phosphopeptide 2 resulting fromryptic digestion of either g-PAK1–96 or g-PAK, under-cores the effectiveness of a reproducible phosphopep-ide mapping system. Further, the high degree of re-roducibility of this method was also confirmed by thenalysis of the phosphorylation status of proteins otherhan g-PAK, including p53 and RAP74 (Gatti, A.,raugh, J. A., and Liu, X., unpublished data).Overall, our data from manual and automated se-

uencing of g-PAK and other phosphoproteins clearlyndicate that the methodology described here to frac-ionate and isolate phosphopeptides does not cause anyetectable N-terminal blockage of the isolated phos-hopeptides.In addition to the advantage of unifying the analyt-

cal and the preparative experimental approaches, thewo-dimensional peptide gel system described hereresents several advantages over other peptide map-ing systems. When compared to the conventional two-imensional separation of phosphopeptides on thin-ayer cellulose plates (17), the fact that our peptideeparation is entirely run on polyacrylamide gelslearly simplifies the processing of the isolated phos-hopeptides for preparative purposes. As pointed outn previous studies (18, 19), the use of two-dimensionalhosphopeptide mapping on thin-layer cellulose platesor purposes other than analytical may be problematicue to the lack of an accurate recovery of certain phos-hopeptides. In addition, tryptic phosphopeptide mapsypically resulting from conventional two-dimensionaleptide analysis on thin-layer cellulose plates areomewhat less focused than those generated by oureptide gel system. Such an improved resolutionlearly represents an important advantage for the an-lytical approach. Moreover, the sensitivity of thisethodological approach is such that excellent phos-

hopeptide maps can be obtained with a few hundred

IG. 6. Identification of the major phosphorylation site in the se-uence of g-PAK1–96. The sequence of g-PAK1–96, as deduced from theorresponding cDNA (4), is shown. The tryptic site involved in theeneration of phosphopeptide 2 is indicated by an arrow, while thehosphorylated residue is underlined.

isintegrations per minute of 32P-labeled peptides. p

On the grounds of the previously described charac-eristics of the high-reticulation alkaline PAGE (2), oureptide gel system allows an approximate evaluation ofhe size of the phosphopeptides, with the smallest onesigrating farthest, which is particularly useful when

electing phosphopeptides for sequencing analysis.Further, the first-dimensional isoelectric focusing is

xpected to enable the resolution of different phosphor-lation states of the same peptide. This is to be com-ared to the reversed-phase HPLC-based method toesolve cognate phosphopeptides, which is dependentn the peptide as well as on the chromatography con-itions (20, 21).On the other hand, reversed-phase HPLC usually

llows very good recovery, yielding a clean, salt-freeample that can be directly subjected to automatedequencing and/or mass spectrometry analysis. How-ver, in case of a complex peptide mixture, such as thatesulting from tryptic digestion, the subsequent anal-sis of individual peaks is often hindered by the pres-nce of multiple peptides in individual HPLC fractions18). The peptide gel system here described, beingased on the combination of two dimensions, facilitateshe analysis of complex mixtures that would remainnresolved by one-dimensional procedures. Given thedvantages of reversed-phase chromatography, weade use of C18 cartridges to carry out a simple

leanup of the isolated phosphopeptides prior to se-uencing analysis. With regard to the potential loss ofmall phosphopeptides during this cleanup step, itust be noted that peptides whose size is too small to

e retained by the C18 resin are also characterized byneffective binding to sequencing supports, includingequelon AA (data not shown).The possibility that some phosphopeptides do not

nter one or the other gel dimension of our system orhat they leak out during the protocol represents aotential reason for concern. However, the successfuldentification of multiple autophosphorylation sites in-PAK, to be reported elsewhere (Gatti, A., Huang, Z.,uazon, P. T., and Traugh, J. A., submitted for publi-ation), argues against the relevance of such a concernnd underscores the effectiveness of our experimentalpproach.Most importantly, our method is simple, inexpen-

ive, and does not require any dedicated apparatus toenerate reproducible and high-resolution phos-hopeptide maps. Because of these features, we antic-pate this two-dimensional peptide gel system beingarticularly advantageous in those instances such asn vivo phosphorylation studies when a limitingmount of starting phosphoprotein and/or a low stoi-hiometry of protein phosphorylation hinder the possi-ility of directly sequencing the phosphopeptides. Thiss the case when the identification of the exact sites of

rotein phosphorylation depends on the need to narrow

dttpiT

sntapmcoutpl

A

rfTg(

R

1

1

11

1

1

1

1

1

1

2

204 GATTI AND TRAUGH

own the list of candidate sites by subjecting the ini-ially fractionated phosphopeptides to secondary diges-ion with site-specific proteases or chemical agentsrior to phosphopeptide mapping and manual sequenc-ng of the resulting phosphopeptides (Gatti, A.,raugh, J. A., and Liu, X., unpublished data).In summary, we have shown that a novel two-dimen-

ional peptide gel system provides an excellent alter-ative to the conventional phosphopeptide mappingechniques currently in use. Being suitable for bothnalytical (comparative phosphopeptide mapping) andreparative (amino acid sequencing) purposes, thisethod should prove useful in the broad range of bio-

hemical studies focused on the phosphorylation statusf proteins. Moreover, since a similar method can besed to fingerprint any radiolabeled or fluorescent pro-ein, it may potentially be useful in the analysis ofosttranslational modifications other than phosphory-ation.

CKNOWLEDGMENTS

We thank Zhongdong Huang and Barbara Walter for providingecombinant g-PAK1–96 and g-PAK, respectively. Also thanks to Pro-essor Wing Tai Cheung for important technical advice and to Dr.im Kingan for the microsequencing. This study was supported byrants from the U.S. Public Health Service (GM26738) and NSFBIR-9601810).

EFERENCES

1. Hardie, G., Campbell, D. G., Caudwell, B. F., and Haystead,T. A. J. (1993) in Protein Phosphorylation, a Practical Approach(Hardie, D. G., Ed.), pp. 61–85. IRL Press, Oxford.

2. West, M. H. P., Wu, R. S., and Bonner, W. M. (1984) Electro-phoresis 5, 133–138.

3. Lim, L., Manser, E., Leung, T., and Hall, C. (1996) Eur. J. Bio-chem. 242, 171–185.

2

4. Jakobi, R., Chen, C. J., Tuazon, P. T., and Traugh, J. A. (1996)J. Biol. Chem. 271, 6206–6211.

5. Sells, M. A., and Chernoff, J. (1997) Trends Cell Biol. 7, 162–167.

6. Manser, E., Huang, H. Y., Loo, T. H., Chen, X. Q., Dong, J. M.,Leung, T., and Lim, L. (1997) Mol. Cell. Biol. 17, 1129–1143.

7. Gatti, A., and Robinson, P. J. (1997) Eur. J. Biochem. 249, 92–97.

8. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Sci.USA 76, 4350–4354.

9. Lui, M., Tempst, P., and Erdjument-Bromage, H. (1996) Anal.Biochem. 241, 156–166.

0. Lopez, M. F., Barry, P., Sawlivich, W. B., Hines, T., and Skea,W. M. (1994) Appl. Theor. Electrophoresis 4, 95–102.

1. Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166,368–379.

2. Sullivan, S., and Wong, T. W. (1991) Anal. Biochem. 197, 65–68.3. Dedner, N., Meyer, H. E., Ashton, C., and Wildner, G. F. (1988)

FEBS Lett. 236, 77–82.4. Ikebe, M., and Reardon, S. (1990) J. Biol. Chem. 265, 8975–

8978.5. Soula, M., Rothhut, B., Camoin, L., Guillaume, J. L., Strosberg,

D., Vorherr, T., Burn, P., Meggio, F., Fisher, S., and Fagard, R.(1993) J. Biol. Chem. 268, 27420–27427.

6. Zhang, Y., Beck, C. A., Poletti, A., Edwards, D. P., and Weigel,N. J. (1994) J. Biol. Chem. 269, 31034–31040.

7. Boyle, W., der Geer, P. V., and Hunter, T. (1991) Methods Enzy-mol. 201, 110–149.

8. de Carvahlo, M. G. S., McCormack, A. L., Olson, E., Ghomashchi,F., Gelb, M. H., Yates, J. R., and Leslie, C. C. (1996) J. Biol.Chem. 271, 6987–6997.

9. Borsch-Haubold, A. G., Bartoli, F., Aselin, J., Dudler, T.,Kramer, R. M., Apitz-Castro, R., Watson, S. P., and Gelb, M. H.(1998) J. Biol. Chem. 273, 4449–4458.

0. Fiol, C. J., Mahrenholz, A. M., Wang, Y., Roeske, R. W., andRoach, P. J. (1987) J. Biol. Chem. 262, 14042–14048.

1. Amess, B., Manjarrez-Hernandez, H. A., Howell, S. A., Lear-month, M., and Aitken, A. (1992) FEBS Lett. 297, 285–291.