ANALYTICAL

Analytical Biochemistry 321 (2003) 252–255

BIOCHEMISTRY

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Notes & Tips

A cleanup step maximizes the immunoprecipitation oftyrosine-phosphorylated peptides by a conventional

antiphosphotyrosine antibody

Andrea Gatti*

Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA

Received 13 May 2003

In the effort to increase the efficiency by which anti-

phosphotyrosine (anti-P-Tyr)1 antibodies can select ty-rosine-phosphorylated peptides out of protein digests, it

is reported here that a preliminary Sep-Pak-C18 car-

tridge (C18)-based cleanup step greatly potentiates the

capacity of a conventional anti-P-Tyr antibody to im-

munoprecipitate the target peptide of c-Src-phosphory-

lated enolase.

An aberrant activation of tyrosine kinases is a typical

effect of oncogene signaling, thereby representing ahallmark of several types of cancer. In fact, the analysis

of the global tyrosine kinase activity (i.e., phosphoty-

roproteome) is an important aspect of disease pheno-

typing [1,2]. The possibility of using an anti-P-Tyr

antibody to immunoprecipitate tyrosine-phosphorylated

proteins [3] and peptides [4,5] is of great interest, because

such a procedure is easy to standardize (by using the

same antibody) and is unbiased (not requiring a pre-liminary selection of candidate substrates).

An efficient recovery of tyrosine-phosphorylated tar-

gets via anti-P-Tyr immunoprecipitation remains an

elusive goal, particularly when dealing with a limited

amount of starting material. As recent methods in the

identification of substrates of protein kinases have been

mostly based on the analysis of constituent peptides [6],

the present study is focused on the possibility of select-ing tyrosine-phosphorylated peptides out of protein

digests.

* Corresponding author. Present address: Via Stringher 27, Rome

00191, Italy. Fax: 1-39-06-36300832.

E-mail address: andreagatti4@libero.it.1 Abbreviations used: C18, Sep-Pak-C18 cartridge; MS, mass

spectrometry; PAGE, polyacrylamide gel electrophoresis; P-Tyr,

phosphotyrosine; TFA, trifluoroacetic acid.

0003-2697/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/S0003-2697(03)00457-3

Methods

Preparation of 32P-labeled peptides. Liquid-phase

phosphorylation was carried out in a 50-ll reaction

volume in 20mM Tris–HCl, 10mM MgCl2, 1mM

dithiothreitol, pH 7.4, in the presence of 0.2mM [c-32P]ATP (1000 cpm/pmol), 50 lg of purified Abl peptide

substrate (New England Biolabs), and 500 units of pu-

rified Abl protein tyrosine kinase (New England Bio-

labs). After 30min of incubation at 30 �C, the reactionwas terminated by adding excesses of EDTA and unla-

beled ATP, prior to 1:3 dilution with a buffer consisting

of 50mM NH4HCO3 and 1% Zwittergent 3-16.

Enolase from rabbit muscle (Sigma) was immobilized

on nitrocellulose (0.45 lm, Schleicher & Schuell) by

spotting a 10-ll aliquot (25 lg) onto nitrocellulose

(0.3� 0.3 cm) before air drying. Solid-phase phosphor-

ylation was carried out in 0.15ml of 20mM Tris–HCl,10mM MgCl2, 1mM dithiothreitol, pH 7.4, in the

presence of 0.2mM [c-32P]ATP (1000 cpm/pmol),

75 units of c-Src (Upstate Biotechnology), and nitrocel-

lulose-bound enolase. After 30min of incubation at

30 �C, the reaction was terminated by extensive washing

of the nitrocellulose.

Proteolytic digestion and cleanup. After radiolabeling,

the nitrocellulose-bound enolase was proteolyticallycleaved upon incubation in 0.15ml of 50mM

NH4HCO3, 1% Zwittergent 3-16, and 50 ng/ll of trypsinfor 24 h at 30 �C. When indicated, tryptic digests were

subjected to a C18-based cleanup [7] prior to anti-P-Tyr

immunoprecipitation. Briefly, the sample was loaded

onto a preequilibrated C18 cartridge (Sep-Pak car-

tridges, 50mg, Waters), which was then washed with 10

volumes of 0.1% trifluoroacetic acid (TFA). Phospho-peptides were eluted in 66% of acetonitrile plus 0.1%

Fig. 1. Gel electrophoresis of the 32P-labeled Abl peptide. The in vitro

phosphorylation of the Abl peptide substrate was carried out under

liquid-phase conditions, prior to incubation with the agarose-conju-

gated anti-P-Tyr antibody. Equivalent fractions (5–10% of total) of the

kinase assay mix, the anti-P-Tyr immunoprecipitate, and the respective

supernatant fraction (unbound to the anti-P-Tyr antibody) were elec-

trophoresed onto the 40% peptide PAGE before (top) or after (bot-

Notes & Tips / Analytical Biochemistry 321 (2003) 252–255 253

TFA, taken to dryness in a speed-vac, and reconstitutedin 0.15ml of 50mM NH4HCO3 and 1% Zwittergent

3-16.

Immunoprecipitation and visualization of peptides.

The 32P-labeled peptides were immunoprecipitated with

the agarose-conjugated anti-P-Tyr antibody (4G10; S.

Cruz). In particular, a 15-ll aliquot of beads of 4G10

was incubated with 0.15ml of reaction volume under

rotation at 4 �C. Beads were washed twice with ice-cold20mM Tris–HCl, 1mM sodium orthovanadate, pH

7.4, and finally incubated with 0.15ml of 0.5M NaOH

for 10min to elute the 32P-labeled peptides. Aliquots

(15 ll) of the neutralized immunoprecipitates were

electrophoresed on a 40% polyacrylamide alkaline slab

gel (40% peptide PAGE), as previously described [7].

When indicated, samples were subjected to a C18-

based cleanup [7] prior to the 40% peptide PAGE.After electrophoresis, the 32P-labeled peptides were vi-

sualized upon autoradiography of the dried peptide gel

and Cerenkov-counted upon excision of relevant gel

bands.

tom) being subjected to a C18-based cleanup step. The relevant

portions of the autoradiograms of dried 40% gels are shown. The data

shown are representative of two independent experiments.

Results and discussion

Initially, the possibility of using a conventional anti-

P-Tyr antibody (4G10) to immunoprecipitate the tyro-

sine-phosphorylated peptides out of a simple mixture

was examined. To this aim, the simplest experimental

model consists of a purified peptide substrate being

preliminarily phosphorylated by the corresponding ty-

rosine kinase. Here, the Abl peptide was incubated in

presence of [32P]ATP with the tyrosine kinase Abl in aconventional in vitro kinase assay (i.e., under liquid-

phase conditions). The 32P-labeled peptide was then

immunoprecipitated with the agarose-conjugated 4G10,

eluted from the agarose beads, and visualized via 40%

peptide PAGE [7]. Under these conditions (Fig. 1, top),

slightly more 32P-labeled peptide was detected in the

anti-P-Tyr immunoprecipitate than what was lost in the

supernatant of the immune reaction. Significantly, asimilar outcome was obtained when the above samples

were subjected to a C18-based cleanup step prior to 40%

peptide PAGE (Fig. 1, bottom).

Having verified the capacity of 4G10 to select for ty-

rosine-phosphorylated peptides out of a simple reaction

mix, a similar approach was then applied to a sample as

complex as the tryptic digest of a c-Src-phosphorylated

protein. Similar to previous studies [8,9], a nitrocellulose-immobilized substrate was employed as target of the

soluble protein kinase (i.e., solid-phase kinase assay) to

facilitate the overall flow of operations prior to solid-

phase tryptic digestion of the substrate (a condition

known to maximize the proteolytic cleavage of most

substrates). Note that the specificity and the efficiency of

the in vitro substrate phosphorylation under solid-phase

and liquid-phase conditions were previously shown to be

indistinguishable [9].

The solid-phase kinase assay is believed to circumventsome of the typical problems of more conventional as-

says. In fact, an efficient phosphorylation of enolase by

c-Src under liquid-phase conditions is exclusively ac-

complished if the substrate is preincubated in acidic

buffer [10]. In line with the view that a partially dena-

tured enolase serves as best substrate of c-Src in vitro,

the efficiency by which c-Src phosphorylates enolase

under solid-phase conditions (data not shown) mightreflect the occurrence of protein unfolding during im-

mobilization on nitrocellulose.

To assess the capacity of 4G10 to immunoprecipitate

the tyrosine-phosphorylated peptides generated upon

solid-phase proteolysis of c-Src-phosphorylated enolase,

the tryptic digest of enolase was subjected to anti-P-Tyr

immunoprecipitation prior to peptide fractionation and

visualization on 40% peptide PAGE. Surprisingly, theamount of 32P-labeled peptides recovered in the anti-P-

Tyr immunoprecipitate was negligible (data not shown).

In this regard, it is worth noting that a cleanup step (i.e.,

affinity chromatography) had been carried out prior to

the anti-P-Tyr immunoprecipitation in one of the few

studies documenting the selection of tyrosine-phos-

phorylated peptides out of protein digests [5]. Such

consideration, taken together with the finding that theC18-based cleanup step did not interfere with the overall

recovery of the radiolabeled Abl peptide (Fig. 1),

prompted the idea of examining whether a C18-based

cleanup of the tryptic digest of c-Src-phosphorylated

254 Notes & Tips / Analytical Biochemistry 321 (2003) 252–255

enolase might facilitate the immunoprecipitation of thetyrosine-phosphorylated phosphopeptides.

As shown in Fig. 2, the inclusion of the C18-based

cleanup step prior to the anti-P-Tyr immunoprecipita-

tion enabled an efficient recovery of the 32P-labeled

peptide out of the tryptic digest of c-Src-phosphorylated

enolase. More than 50% of the original amount of such

peptide was routinely recovered in the anti-P-Tyr im-

munoprecipitate, provided that the C18-based cleanupwas carried out. On the other hand, the amount of 32P-

labeled peptide recovered upon direct anti-P-Tyr im-

munoprecipitation (with no inclusion of the C18-based

step) was negligible. Conversely, the 32P-labeled peptide

detected in the supernatant fractions (not bound to the

anti-P-Tyr antibody) of the above immunoreactions was

negligible or abundant, depending on whether the

cleanup step was included or not.The molecular mechanism underlying the effect of the

C18-based cleanup on the immunoprecipitation of ty-

rosine-phosphorylated peptides is currently unclear: it

may relate to a change either in the peptide conforma-

tion (as a consequence of using acetonitrile in the elution

from C18) or in the sample buffer (due to the removal

of factors interfering with the action of the 4610).

Fig. 2. Gel electrophoresis of the 32P-labeled peptide from c-Src-

phosphorylated enolase. Both in vitro phosphorylation and proteolysis

of enolase were carried out under solid-phase conditions. Tryptic di-

gests were either directly incubated with the anti-P-Tyr antibody or

subjected to the C18-based cleanup step prior to the anti-P-Tyr

immunoprecipitation. Equivalent fractions (10% of total) of the C18-

eluted tryptic digest, the respective immunoprecipitate, and the su-

pernatant (unbound to the anti-P-Tyr antibody) were electrophoresed

onto the 40% peptide PAGE. The arrowhead indicates the migration

of an arbitrary amount of free radiolabel. When expressing the amount

of 32P-labeled peptide being recovered upon anti-P-Tyr immunopre-

cipitation of the C18-eluted tryptic digest compared to that contained

in an equivalent fraction of the original tryptic digest, the mean va-

lue�SE of three independent experiments is 52.7� 6.6.

However, the finding that the C18-based cleanup steppermitted an efficient immunoprecipitation of the 32P-

labeled peptide of c-Src-phosphorylated enolase (Fig. 2)

and caused no significant loss of the immunoprecipi-

tated Abl peptide (Fig. 1) is indicative of a general ap-

plicability of the method here presented.

The present study has not addressed the issue of how

to identify the individual phosphopeptide of c-Src-

phosphorylated enolase that is enriched upon the use ofthe described protocol. Obviously, the recent progres-

sion in the technology of mass spectrometry (MS)

should be taken into account when aiming at such a

goal. Tyrosine phosphorylation is often substoichio-

metric, such that the tyrosine-phosphorylated peptides

are present in lower abundance than their unphos-

phorylated counterparts. The attempt to identify the

tyrosine-phosphorylated peptides by MS and ultimatelygenerate profiles of phosphotyroproteome is believed to

become easier upon enrichment of the relevant se-

quences [6]. In the present study, the enrichment step is

based on a C18-based cleanup to be carried out after

proteolysis of the tyrosine-phosphorylated substrate and

before anti-P-Tyr immunoprecipitation.

Given the relevance of tyrosine phosphorylation

within the signal transduction of multicellular organ-isms, the signaling state of given cells may be monitored

by assessing changes in the phosphorylation of relevant

substrates at specifically targeted tyrosine sites. The

presently described procedure, to be possibly combined

with an appropriate protein array, is expected to facili-

tate the comprehensive characterization of the global

state of tyrosine phosphorylation and contribute to the

scope of disease phenotyping.

Acknowledgment

The experimental work was carried out in the labo-

ratory of Dr. Moses Chao, who kindly provided finan-

cial support for this project.

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