RESEARCH ARTICLE
© 2002 Nature Publishing Group http://biotech.nature.com
Proteomic profiling of mechanistically distinct
enzyme classes using a common chemotype
Gregory C. Adam1, Erik J. Sorensen1, and Benjamin F. Cravatt1,2*
Published online: 1 July 2002, doi:10.1038/nbt714
Proteomics research requires methods to characterize the expression and function of proteins in complex
mixtures. Toward this end, chemical probes that incorporate known affinity labeling agents have facilitated the
activity-based profiling of certain enzyme families. To accelerate the discovery of proteomics probes for
enzyme classes lacking cognate affinity labels, we describe here a combinatorial strategy. Members of a
probe library bearing a sulfonate ester chemotype were screened against complex proteomes for activitydependent protein reactivity, resulting in the labeling of at least six mechanistically distinct enzyme classes.
Surprisingly, none of these enzymes represented targets of previously described proteomics probes. The sulfonate library was used to identify an omega-class glutathione S-transferase whose activity was upregulated
in invasive human breast cancer lines. These results indicate that activity-based probes compatible with
whole-proteome analysis can be developed for numerous enzyme classes and applied to identify enzymes
associated with discrete pathological states.
In the postgenomic era, biological researchers face the unprecedented challenge of assigning molecular and cellular functions to
each of the >30,000 protein products encoded by the human
genome. Proteomics aims to accelerate this process by developing
and applying methods for the parallel analysis of large numbers of
proteins1,2. Proteomics approaches generally fall into two complementary categories: methods for the global analysis of protein
expression and methods for the global analysis of protein function.
To date, large-scale efforts to characterize protein expression have
mostly relied on two-dimensional electrophoresis (2DE), protein
staining, and mass spectrometry (MS) as separation, detection, and
identification methods, respectively3. Methods for 2DE-MS permit
the consolidated analysis of the relative abundance and modification state of numerous proteins from endogenous sources, thereby
facilitating the identification of protein markers of discrete physiological and pathological states4. However, several important protein classes, including low-abundance and membrane proteins,
remain difficult to analyze by 2DE3,5,6. Additionally, because standard 2DE-MS methods focus on measurements of protein abundance, they provide only an indirect estimate of protein function
and may fail to detect important post-translational forms of regulation such as those mediated by protein–protein and/or
protein–small molecule interactions.
To expedite the analysis of protein function, several methods have
been developed to characterize protein activities on a global scale.
These include large-scale yeast two-hybrid screens7,8, which aim to
construct a comprehensive map of protein–protein interactions that
occur in the cell, and protein arrays9,10, which aim to provide an assay
platform to rapidly assess the function of recombinantly expressed
proteins. Although these methods have the advantage of assigning
specific molecular activities to individual protein products, they also
rely on recombinantly expressed proteins studied in artificial envi-
ronments, and therefore do not directly assess the functional state of
these biomolecules in their natural settings.
Recently, a third strategy for proteome analysis has emerged that
uses chemical probes to profile the activity of enzyme superfamilies
in complex proteomes11. These probes are composed of at least two
general molecular elements: a reactive group that binds and covalently modifies the active sites of many members of a given enzyme
class, and a chemical tag for the detection and isolation of reactive
enzymes. Because these probes label their target proteins on the basis
of functional properties rather than abundance, they provide a sensitive readout of changes in enzyme activity that occur in complex
proteomes, distinguishing, for example, active proteases from their
inactive zymogen and/or inhibitor-bound forms12. Additionally,
active site–directed proteomics probes can be used in competitive
binding assays as a rapid and consolidated screen to identify selective
small-molecule inhibitors of individual members of large enzyme
classes12,13. Thus, for the applicable enzyme families, chemical proteomics methods offer a means to monitor changes in the activity of
enzymes in native proteomes, while at the same time facilitating the
downstream functional characterization of these proteins.
To date, most activity-based proteomics probes have incorporated
well-known affinity-labeling agents as their reactive groups. Thus, in
the design of these probes, researchers have capitalized on a rich history of mechanistic studies associated with particular classes of
enzymes to create chemical probes with predictable proteome reactivities12–15. For many enzyme classes, however, cognate affinity labels
do not yet exist, thus limiting the design of activity-based proteomics
probes. To expand the number of enzyme families susceptible to
analysis by activity-based profiling methods, we have adopted a
nondirected or combinatorial strategy in which libraries of candidate probes are screened against complex proteomes for activitydependent protein reactivity. We recently described the synthesis and
The Skaggs Institute for Chemical Biology and Departments of 1Chemistry and 2Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road,
La Jolla, CA 92037. *Corresponding author (cravatt@scripps.edu).
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Soluble and membrane proteomes
derived from a variety of mouse tisO
R=
N
sues were treated with the library of
O
rhodamine-tagged sulfonate probes,
Phenyl
Octyl
Quinoline
resulting in the detection of numerO
O
O
ous heat-sensitive protein reactivities
H3C
R S O
R S O
NO2
N
(Fig. 2). Although several proteins
O
O
H
O
O
N
O
were labeled by multiple members of
S
Nitrophenyl
Naphthyl
Mesyl
NH
N
N
the sulfonate library (Fig. 2A, arrowH
H
H
S
HN
NH
heads), others exhibited strong selecO
N
O OH
O
tivity for a single sulfonate probe
Rhodamine-tagged
Biotinylated
(Fig. 2A, asterisk). Conversely, some
Pyridyl
Thiophene
N
sulfonate esters
sulfonate esters
sulfonate probes labeled numerous
proteins (such as phenyl), whereas
Figure 1. Synthesis of rhodamine- and biotin-tagged sulfonate-ester probe libraries.
others displayed more restricted proteome reactivities (such as octyl). On
preliminary evaluation of a probe library bearing a sulfonate ester
the basis of these initial profiles, the phenyl sulfonate probe was
reactive group coupled to a variable alkyl/aryl-binding group16.
selected for a more detailed comparative analysis of mouse tissue
Members of this probe library were found to label a class I aldehyde
proteomes. Most of the proteins labeled by the phenyl sulfonate
dehydrogenase (ALDH-1) in an active site–directed manner.
probe exhibited a restricted distribution, often appearing in only
However, additional sulfonate-labeled proteins were not identified in
one or two of the tissues examined (Fig. 2B). These data suggested
this study, leaving a central question unanswered: are probes founded
that targets of the sulfonate library did not represent broadly
on a sulfonate chemotype restricted to labeling ALDH enzymes, or
expressed “housekeeping” proteins.
alternatively, might they serve as activity-based profiling agents for
To gain further insight into the types of proteins displaying
additional classes of enzymes? We now show that members of this sulactivity-based reactivity with the sulfonate library, we evaluated
fonate library label at least six mechanistically distinct enzyme classes
the effects of various enzyme cofactors and related additives on
in complex proteomes and provide evidence that these labeling events
the phenyl sulfonate–labeling profile of the heart soluble
occur in the active sites of the targeted enzymes. Moreover, we
proteome. Several enzyme cofactors were found to influence the
demonstrate how the sulfonate probe library can be used to both
sulfonate reactivity of specific heart proteins. For example, the
identify active enzymes differentially expressed in complex proteomes
addition of nicotinamide-adenine dinucleotide phosphate
and provide insights into the functional properties of these proteins.
(NADP)+ and NAD+ was found to strongly inhibit the sulfonate
reactivity of 36 kDa and 55 kDa proteins, respectively (Fig. 2C,
Results
arrowheads). In contrast, both NADP+ and coenzyme A (CoA)
Activity-based screening of proteomes with fluorescent sulfonate
caused an increase in the labeling of a pair of 60–65 kDa proteins.
ester probes. Our original library of biotin-tagged sulfonate ester
These data suggest that several of the sulfonate labeling events
probes was used in combination with avidin-based chemiluminesoccurred in the active sites of cofactor-dependent enzymes, and,
cence blotting techniques to screen complex proteomes for activitydepending on the enzyme, the addition of cofactor either
based protein labeling16, where activity-based labeling events were
enhanced or inhibited sulfonate labeling.
defined as those that occurred with
native but not heat-denatured proA
C
B
teomes. Although these initial studies
resulted in the identification of a class
I ALDH as an activity-dependent sulfonate target, the discovery of additional sulfonate-labeled proteins was
hampered by the limited sensitivity
and throughput of biotin–avidin
screening methods. To address these
limitations, we conjugated the sulfonate ester library to a rhodamine
derivative (Fig. 1), permitting the use
of in-gel fluorescence scanning as a
rapid, quantitative, and sensitive
screen for activity-based protein labeling events17. Thus, we describe here a
two-tiered proteome screening platform in which, first, rhodamineFigure 2. Profiling complex proteomes with a rhodamine-tagged sulfonate probe library. (A) Labeling profiles
tagged sulfonate probes are used to for eight members of the sulfonate probe library reacted with the mouse heart soluble proteome. Arrowheads
generate activity-based profiles of and asterisk highlight examples of heat-sensitive protein targets that display broad and selective sulfonatecomplex proteomes, and then the cor- reactivity profiles, respectively. ∆, Heat-denatured proteome. (B) Phenyl sulfonate–labeling profiles of soluble
responding biotinylated probes are and membrane fractions of mouse tissue proteomes, highlighting heat-sensitive protein targets that display
broad (arrowheads) and restricted (asterisks) tissue distributions. (C) The effect of cofactors and additives
applied for the affinity isolation and (500 µM) on the sulfonate-reactivity profile of the mouse heart soluble proteome, highlighting proteins with
identification of proteins that exhibit sulfonate reactivities that are decreased (arrowheads) and increased (asterisks) by the presence of cofactors.
activity-dependent sulfonate labeling. Lower panel represents a reduced-intensity image of the 50 kDa region of the upper panel.
© 2002 Nature Publishing Group http://biotech.nature.com
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RESEARCH ARTICLE
B
C
Identification of protein targets of the A
sulfonate probe library. A biotinylated version of the phenyl sulfonate probe was used
in combination with avidin-based chromatography to affinity isolate heat-sensitive
protein reactivities from complex proteomes (see Experimental Protocol). These
proteins were then digested with trypsin and
identified by mass spectrometry analysis of
the resulting peptide maps (Fig. 3A). For
lower-abundance sulfonate targets (such as
dihydrodiol dehydrogenase (DDH)), proteomes were first fractionated over a Figure 3. Identification of protein targets of the sulfonate probe library. (A) Molecular identities of six
displaying heat-sensitive sulfonate reactivity. These proteins were affinity isolated and
Q-Sepharose anion exchange column to proteins
identified by mass spectrometry analysis (Experimental Protocol). The sulfonate target epoxide
provide enriched samples of these enzymes hydrolase (EH; asterisk) was most effectively visualized in complex proteomes with the biotin-tagged
for subsequent affinity purification. version of the phenyl sulfonate probe (see (B), right panels). (B) Phenyl sulfonate labeling of the
Although we have focused in this study on identified enzymes recombinantly expressed in COS-7 cells. (C) Selective inhibition of phenyl
+
+
the identification of soluble targets of the sulfonate labeling of ALDH-1, DDH and ECH-1 by the cofactors NAD (500 µM), NADP (500 µM),
and CoA (2 mM), respectively.
sulfonate probes, in related efforts, we have
applied similar methods to affinity enrich
and identify membrane-associated serine hydrolase enzymes from
with the biotinylated version of the phenyl sulfonate probe (Fig. 3B,
Triton X-100–solubilized cell membrane fractions using fluoright panels), indicating that for certain proteins, the chemical
rophosphonate (FP) probes (data not shown). These data indicate
structure of the detection tag can influence probe reactivity.
that both soluble and membrane-associated targets of chemical
Although heat-sensitive probe reactivity represents a simple and
proteomics probes can be identified using a combination of avidineffective primary screen for activity-based protein labeling events,
based enrichment and mass spectrometry procedures.
we sought additional biochemical evidence that sulfonate probes
Notably, the identified protein targets of the phenyl sulfonate
were modifying the active sites of the targeted enzymes. For examprobe represented enzymes from several distinct mechanistic classple, a subset of the sulfonate targets represented cofactor-dependent
es: one epoxide hydrolase (cytoplasmic EH), two ALDHs (ALDH-1
enzymes, and for these proteins, the addition of excess cofactor was
and ALDH-7), one thiolase (acetyl CoA acetyltransferase), one
found to inhibit sulfonate labeling (Fig. 3C). Interestingly, these
NAD/NADP-dependent oxidoreductase (DDH), and one enoyl
competition experiments recapitulated the established cofactorcoA hydratase (peroxisomal ECH-1). To confirm that database
binding selectivities of the targeted enzymes, with the sulfonate
searches had correctly identified protein targets of the sulfonate
labeling of ALDH-1 and DDH being exclusively blocked by NAD+
and NADP+, respectively18,19. In hindsight, the sensitivity of sulfonate
library, cDNAs corresponding to these enzymes were isolated and
labeling to the presence of cofactor had been detected for both
transiently transfected into COS-7 cells. A comparison of the labelenzymes in native proteomes (Fig. 2C), suggesting that activitying profiles of transfected COS-7 cells confirmed the reactivity of
based probes can be used to predict the cofactor dependence of their
each enzyme with the rhodamine-coupled phenyl sulfonate probe
protein targets even before target identification. Addition of the
(Fig. 3B). Notably, however, one enzyme, EH, reacted more strongly
substrate analog CoA was found to substantially
A
B
reduce labeling of ECH-1 without affecting the sulfonate reactivity of the CoA-independent enzyme
ALDH (Fig. 3C, lower panels). Collectively, these
results indicate that several mechanistically distinct
enzyme classes are targeted in an active site–directed
manner by chemical probes that incorporate a sulfonate reactive group.
Comparative activity-based profiling of human
breast cancer cell lines. Sulfonate probes were used to
screen for enzyme activities differentially expressed
C
across a panel of estrogen receptor–positive (ER+)
and –negative (ER–) human breast cancer cell lines.
For breast carcinomas, a strong inverse correlation
exists between ER expression and several metastatic
phenotypes, including cell motility and invasiveness20,
and therefore enzymes upregulated in ER– breast cancer lines represent candidate biomarkers and theraFigure 4. Profiling proteomes of human breast cancer cell lines with the sulfonate probe
peutic targets for aggressive forms of breast cancer.
library. (A) Phenyl sulfonate labeling profiles for two ER+ (MCF7, T-47D) and two ER–
We detected several proteins with heat-sensitive
(MDA-MB-231, MDA-MB-435) breast cancer lines. Arrowheads designate heat-sensitive
phenyl sulfonate reactivity in ER+ and ER– breast canprotein targets. (B) Quantitation of GSTO 1-1 activity expressed by human breast cancer
lines as measured by phenyl sulfonate–labeling intensities (arbitrary units; n = 4 per cell
cer lines (Fig. 4A, arrowheads). Most notably, we
line; P < 0.01, planned comparisons). Pretreatment with the GST substrate glutathione
identified a 30 kDa sulfonate target that was strongly
(1 mM, 10 min) inhibited >95% of the phenyl sulfonate labeling of the GSTO1-1 enzyme
upregulated in both ER– breast cancer lines MDAfrom either MDA-MB-435 or MDA-MB-231 proteomes (two rightmost bars). (C) Phenyl
MB-231 and MDA-MB-435 relative to the ER+ lines
sulfonate labeling of GSTO 1-1 recombinantly expressed in COS-7 cells. The labeling of
GSTO 1-1, but not ALDH-1, was inhibited by the addition of glutathione.
MCF7 and T-47D. Affinity isolation and mass spechttp://biotech.nature.com
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subclasses of cysteine proteases13,15.
The complementary proteomeEnzyme
Enzyme class
Proteome source
reactivity profiles of the sulfonate
library and other chemical proAcetyl CoA acetyltransferase
Thiolase
Mouse heart
teomics probes suggested that the
Aldehyde dehydrogenase 1
Aldehyde dehydrogenase
Mouse heart
value of these reagents might be
Aldehyde dehydrogenase 7
Aldehyde dehydrogenase
Mouse heart
Dihydrodiol dehydrogenase
NAD/NADP-dependent oxidoreductase
Mouse heart
enhanced by their use in combinaEnoyl CoA hydratase 1, peroxisomal Enoyl CoA hydratase
Mouse heart, human MDA- tion (multiplexing). However, initial
MB-435 cells
attempts to apply mixtures of rhoEpoxide hydrolase, cytoplasmic
Epoxide hydrolase
Mouse heart
damine-tagged sulfonate and fluoGSTO 1-1
Glutathione S-transferase
Human MDA-MB-435 cells
rophosphonate (serinehydrolase–
directed) probes to proteomes of
trometry analysis of this enzyme from MDA-MB-435 cells identihuman breast cancer lines resulted in significant overlap in activified it as an omega-class glutathione S-transferase (GSTO 1-1)21, a
ty-based profiles and therefore poor resolution of the targeted
protein with no previous link to breast cancer. Measurement of the
proteins (data not shown). In contrast, multiplexing rhodamineintegrated band intensity for sulfonate-labeled GSTO 1-1 across
tagged sulfonate probes with a fluorescein-tagged FP probe prothe human breast cancer lines revealed that this enzyme was more
vided an effective means to detect, in a single proteomic sample,
than tenfold upregulated in both ER– lines relative to either ER+
enzyme activities labeled by each class of probe (Fig. 5). These
line (Fig. 4B). The sulfonate reactivity of recombinantly expressed
findings indicate that both spatial and spectral methods can be
GSTO 1-1 was strongly inhibited by the GST substrate glutathione
used to resolve differentially expressed enzyme activities, permit(Fig. 4C), indicating that the sulfonate-GSTO 1-1 reaction
ting the application of mixtures of chemical probes that possess
occurred in the active site of the enzyme. Notably, the addition of
complementary proteome-reactivity profiles. Such multiplexing
glutathione also blocked the sulfonate labeling of the 30 kDa proof chemical probes may prove of particular value for the analysis
tein expressed by MDA-MB-231 and MBA-MB-435 cells (Fig. 4B),
of proteomic samples of finite quantity.
suggesting that this protein in the MDA-MB-231 line also repreDiscussion
sents GSTO 1-1, rather than a distinct comigrating sulfonate target.
In these studies, we have shown that activity-based chemical probes
To confirm this notion, the 30 kDa MDA-MB-231 sulfonate target
compatible with whole-proteome analysis can be developed for
was affinity isolated and identified by mass spectrometry as GSTO
several enzyme classes. In contrast to previously described chemi1-1. This preliminary analysis of human breast cancer lines with
cal proteomics probes that are highly selective for a single class of
the sulfonate probe library highlights the ability of chemical proenzymes, the sulfonate probe library applied here was found to tarteomics approaches to identify previously unrecognized enzyme
get at least six mechanistically distinct enzyme families. Although it
activities associated with pathological conditions such as breast
remains unclear what structural and/or catalytic properties may be
cancer.
shared by these sulfonate-targeted enzymes, they appear to fall into
Multiplexing probes with complementary proteome reactivity
two general classes: enzymes that use covalent mechanisms for
profiles. Collectively, the sulfonate-labeled enzymes identified in
catalysis and enzymes with noncatalytic nucleophilic residues in
mouse tissues and human cell lines represent several mechanistitheir active sites. The first group of enzymes use either cysteine
cally distinct classes of enzymes (Table 1). Interestingly, however,
(ALDH22, thiolase23, GSTO 1-1; ref. 24) or aspartate (EH25)
none of the sulfonate-labeled proteins were members of enzyme
residues as nucleophiles that form temporary covalent adducts
families targeted by previously described chemical proteomics
with substrates during the course of catalysis. In contrast, enzymes
probes that have been shown to label serine hydrolases12,14,17 and
from the latter group do not use covalent mechanisms for catalysis
(DDH, ECH), but rather appear to share the property of exhibiting
a noncatalytic cysteine residue in their active sites26,27. Whether
these catalytic or noncatalytic active-site residues serve as the site
of labeling by the sulfonate probes remains unknown, but it is
noteworthy that precedent exists for both types of active-site modification in the field of natural products. For example, E-64 (ref.
28) and microcystin29 are natural products that covalently modify
catalytic and noncatalytic cysteine residues in the active sites of
proteases and phosphatases, respectively. More generally, these
observations suggest that a diversity of mechanisms can be exploited for the development of chemical probes that profile enzyme
active sites in complex proteomes.
In summary, perhaps the most striking finding of this study is
that none of the sulfonate-labeled enzymes represented targets of
previously described proteomics probes. This discovery suggests
that continued efforts to expand, by both directed and combinatorial methods, the number of enzyme classes susceptible to profiling
with chemical proteomics probes should result in a panel of
Figure 5. Multiplexing activity-based proteomics probes. Human breast
cancer cell lines were treated with a combination of rhodamine-tagged
reagents that can be used either separately or in combination to
phenyl sulfonate (PhSulf–rhodamine) and fluorescein-tagged
accelerate the discovery of enzyme activities associated with disfluorophosphonate (FP–fluorescein) probes (5 µM of each probe).
crete physiological and/or pathological states. These enzymes may
Comigrating FP–fluorescein and PhSulf–rhodamine targets could be
in turn serve as valuable new biomarkers and/or therapeutic targets
resolved by detecting fluorescent signals with 505 nm and 605 nm
bandpass filters, respectively.
for the diagnosis and treatment of human disease.
© 2002 Nature Publishing Group http://biotech.nature.com
Table 1. Sulfonate-labeled enzymes identified from mouse and human proteomes
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Experimental protocol
© 2002 Nature Publishing Group http://biotech.nature.com
Chemical synthesis of sulfonate ester probes. Synthesis of the rhodaminetagged sulfonate ester library followed a method previously described for
the synthesis of biotin-tagged sulfonate ester probes16, with the exception
that 5-(and 6)carboxytetramethylrhodamine ethylenediamine (Molecular
Probes, Eugene, OR), rather than 5-(biotinamido)-pentylamine, was reacted with the final intermediate.
Tissue sample preparation, labeling, and detection. Mouse tissues were
Dounce-homogenized in Tris buffer (50 mM Tris-HCl buffer, pH 8, 0.32 M
sucrose), and the membrane and soluble fractions were separated by highspeed centrifugation (sequential spins of 22,000g (30 min; pellet = membrane fraction) and 100,000g (60 min; supernatant = soluble fraction)).
The membrane fraction was washed twice and resuspended in Tris buffer
without sucrose. Protein samples (2 mg/ml) were treated with 5 µM rhodamine-tagged sulfonate probe (250 µM stock in dimethyl sulfoxide) and
the reactions were incubated at 25°C for 1 h before quenching with 1 volume of standard 2× SDS–PAGE loading buffer (reducing). Quenched reactions were run on SDS–PAGE (30 µg protein/gel lane) and visualized in gel
using a Hitachi FMBio IIe flatbed laser-induced fluorescence scanner
(MiraiBio, Alameda, CA). Labeled proteins were quantified by measuring
integrated band intensities (normalized for volume).
Enrichment and molecular characterization of sulfonate-reactive proteins. Sulfonate protein targets were affinity isolated using biotinylated
sulfonate probes and avidin agarose beads (Sigma, St. Louis, MO) as
described12,16. For affinity isolations directly from tissue or cell line fractions, ∼4 mg of total protein was used as starting material (equivalent to
∼4 × 107 cells). For lower-abundance sulfonate targets, ∼25 mg of total protein was fractionated by Q chromatography and fractions containing the
desired targets were used for affinity isolation. Affinity-isolated proteins
were separated by SDS–PAGE, excised from the gel, and digested with
trypsin. The resulting peptides were analyzed by a combination of matrixassisted laser-desorption mass spectrometry (Voyager-Elite time-of-flight
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MS instrument, PerSeptive Biosystems, Framingham, MA) and liquid
chromatography–electrospray tandem MS (1100 HPLC (Agilent, Palo
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Cancer cell line preparation. Breast cancer cell lines were grown to 80%
confluency in RPMI-1640 medium (Invitrogen) containing 10% FCS and
harvested, sonicated, and Dounce homogenized in 50 mM Tris-HCl (pH
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labeled as described above.
Acknowledgments
We thank G. Hawkins and M. Humphrey for technical assistance; J. Wu for
assistance with mass spectrometry analysis; and J. Williamson, J. Kelly, and the
Cravatt and Sorensen groups for helpful discussions. This work was supported
by the National Cancer Institute of the National Institutes of Health
(CA87660), the California Breast Cancer Research Program, ActivX
Biosciences, and the Skaggs Institute for Chemical Biology.
Competing interests statement
The authors declare competing financial interests: see the Nature Biotechnology
website (http://biotech.nature.com) for details.
Received 11 February 2002; accepted 15 May 2002
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