© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
REVIEW
Assignment of protein function in the
postgenomic era
Alan Saghatelian & Benjamin F Cravatt
Genome sequencing projects have provided researchers with an unprecedented boon of molecular information that promises to
revolutionize our understanding of life and lead to new treatments of its disorders. However, genome sequences alone offer only
limited insights into the biochemical pathways that determine cell and tissue function. These complex metabolic and signaling
networks are largely mediated by proteins. The vast number of uncharacterized proteins found in prokaryotic and eukaroyotic
systems suggests that our knowledge of cellular biochemistry is far from complete. Here, we highlight a new breed of ‘postgenomic’
methods that aim to assign functions to proteins through the integrated application of chemical and biological techniques.
One of the preeminent challenges facing twenty-first century scientists
is the determination of the molecular, cellular and (patho)physiological
functions for the numerous proteins encoded by eukaryotic and prokaryotic genomes. It is clear that this problem cannot be addressed
by the analysis of genome sequences alone. Indeed, a significant fraction of every genome sequenced to date, from bacterial to human, is
composed of uncharacterized proteins. Even those proteins for which
tentative or apparently robust assignments of activity can be made are
susceptible to reinterpretation as our understanding of the molecular
complexity of life increases. For example, it is now clear that proteins
with highly related sequences can perform different functions in vivo,
and, conversely, proteins may show similar activities while lacking discernible sequence or structural similarity1. The additional realization
that one gene can code for tens if not hundreds of different proteins
as a result of post-transcriptional and post-translational processing
and modification indicates that the number of proteins in need of
characterization vastly exceeds the number of genes in the genome2.
These various issues combine to present a formidable, but exciting, set
of experimental problems for contemporary biological and chemical
researchers, all of which distill down to predominantly a single question: how should we undertake the assignment of protein function in
the postgenomic era?
The biochemical properties of proteins are typically determined
in vitro with purified material. Although this classical ‘test tube biochemistry’ approach has succeeded in explaining the activities of many
proteins, it does suffer some shortcomings. First, proteins do not function in isolation in vivo, but rather as parts of complex metabolic and
signaling networks. Proteins are also regulated by post-translational
mechanisms in vivo, including covalent modification and proteinprotein interactions (Fig. 1). These dynamic events create a context
dependency to the performance of proteins in living systems that can be
The Skaggs Institute for Chemical Biology and Departments of Cell
Biology and Chemistry, The Scripps Research Institute, 10550 North
Torrey Pines Road, La Jolla, California 92037, USA. Correspondence
should be addressed to B.F.C. ([email protected]).
130
difficult, if not impossible, to replicate in vitro. From a methodological
perspective, in vitro studies are biased toward proteins that are more
straightforward to express recombinantly and for which activity assays
are available. As such, many challenging classes of proteins, including
membrane-associated and uncharacterized proteins, are not effectively
addressed by classical methods. Finally, the examination of individual
proteins ‘one at a time’ is an uncomfortably slow process, especially
when confronted with the thousands of proteins in current need of
functional characterization.
The limitations of classical biochemical methods have inspired
the development of tools and technologies that can, in a direct and
systematic way, characterize the activities of proteins in complex biological settings. Among these ‘systems biology’ approaches, genetic
techniques, such as targeted gene disruption (gene ‘knockouts’3) and
RNA interference (RNAi)4, have proven particularly powerful, owing
to their remarkable specificity and generality (Fig. 2). Nonetheless,
genetic methods suffer from a lack of temporal control over protein
activity and are not well suited for dissecting the functions of different protein isoforms. The subject of this review is a distinct breed
of systems biology methods that take advantage of chemistry, often
in combination with genetic, protein biochemistry and analytical
techniques, to tackle the problem of characterizing protein function
on a global scale (Fig. 2). As will be seen, a common trait of these
methods is that they are truly ‘postgenomic’ in the sense that they
require complete genome sequences for optimal performance. Thus,
although the limitations of pure reliance on genome sequences will
emerge as a recurring theme, these comments should not be viewed
as a negative critique of genome sequencing efforts. On the contrary,
the chemical biology approaches described herein provide perhaps
the most profound endorsement of the value of complete genome
sequences, which are enabling us to make experimental inquiries
never before deemed possible.
Genomic methods for the assignment of protein function
Prokaryotic organisms provide perhaps the richest and most diverse
source of metabolic enzymes on the planet. These enzymes often
VOLUME 1 NUMBER 3 AUGUST 2005 NATURE CHEMICAL BIOLOGY
REVIEW
PTMs
Endogenous
inhibitors
No PTMs
No
inhibitors
Assignment of
protein function
OH
HO
HO
O
O
AcHN
O
O P O—
OH
© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
In vivo
Protein-protein
interactions
In vitro
Lack of protein
binding partners
Figure 1 Comparison of the endogenous (in vivo) and exogenous (in vitro)
environments typically experienced by proteins, highlighting several natural
modes for their post-translational regulation.
function as large complexes to produce natural products with remarkable biological activities5. To date, more than 200 prokaryotic genomes
have been fully sequenced, with many more projects under way (http://
www.tigr.org/tigr-scripts/CMR2/CMRHomePage.spl). This rich source
of molecular information has spawned the birth of a new discipline
termed ‘genomic enzymology’ to signify a movement away from singleenzyme phenomenology to the study of these proteins in the context of
entire biological systems1. Genomic enzymology aims to integrate findings from multiple fields of research, including comparative genomics,
structural biology and enzyme kinetics, to determine the functions of
uncharacterized enzymes. This multidisciplinary approach has revealed
several cases where the sequence, structure and function of enzymes
diverge in surprising ways.
Divergent functions from similar sequences. The (β/α)8-(TIM) barrel, which is the most prevalent fold found among enzymes, has provided a fertile subject for postgenomic investigations6. Initiated with
the discovery of two (β/α)8-barrel proteins, mandelate racemase and
muconate lactonizing enzyme, that share conserved structures and
active site functional groups, but catalyze different reactions7, this
enzyme superfamily has since been shown to possess members that
perform a wide range of mechanistically diverse chemical transformations. Thus, (β/α)8-barrel enzymes provide a clear case where gene or
protein sequence alone cannot be used to predict function.
Complete genome sequences have afforded enzymologists the first
opportunity to systematically compare (β/α)8-barrel proteins from
distinct organisms in efforts to characterize their unique functions.
This general strategy of comparative genomics8 is particularly useful
for prokaryotic organisms because their genes are often organized into
operons, defined as a set of adjacent genes all under the transcriptional control of the same operator. Gerlt and colleagues have exploited
‘genomic context’ to predict activities for several uncharacterized
(β/α)8-barrel enzymes. For example, the genes for YcjG and YkfB, two
distantly related homologs of o-succinylbenzoate synthase (OSBS),
were both found in the vicinity of genes involved in murein peptidoglycan metabolism9. Knowledge of this metabolic pathway, coupled
with an understanding of the scope of potential reactions catalyzed by
(β/α)8-barrel enzymes, led the researchers to propose that YcjG and
YkfB are L-Ala-D/L-Glu epimerases (Fig. 3a). Subsequent biochemical
analysis of these proteins confirmed that they have L-Ala-D/L-Glu epimerase activity, representing the first examples of enzymes that catalyze
this reaction. Notably, neither enzyme was able to catalyze the OSBS
NATURE CHEMICAL BIOLOGY VOLUME 1 NUMBER 3 AUGUST 2005
Genomics
Proteomics
Metabolomics
Gene knockouts/RNAi
Genomic enzymology
Chemical profiling
Molecular engineering
Targeted
Untargeted
Genome
sequences
Figure 2 Genome sequences have served as a foundation for the advancement
of global experimental approaches to investigate the function of proteins.
reaction, underscoring the remarkable functional diversity that can
exist among sequence-related proteins.
Structural insights into the plasticity of (β/α)8-barrel proteins have
been gained by a comparison of orotidine 5′-monophosphate decarboxylase (OMPDC) and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), two homologous enzymes that catalyze mechanistically
unrelated reactions (metal-independent and Mg2+-dependent decarboxylations, respectively)10. An overlay of the active site structures of
OMPDC and KGPDC revealed that conserved residues occupy nearly
identical positions but serve distinct roles in catalysis. In OMPDC, these
residues are involved in substrate binding, whereas in KGPDC they
serve as ligands to coordinate a Mg2+ ion required for stabilization
of an enediolate anion intermediate (this intermediate is not formed
in OMPDC-catalyzed decarboxylations). The authors conclude that
OMPDC and KGPDC should be designated as members of a suprafamily to underscore that, even though these enzymes have significant
sequence and structural similarity, they use distinct catalytic mechanisms (Fig. 3b). More generally, these discoveries offer an instructive
warning to those eager to predict the functions of uncharacterized proteins solely on the basis of sequence or structural information.
Examples of homologous enzymes that have disparate activities can
also be found outside the (β/α)8-barrel family of enzymes. Whitman and
colleagues have shown that different members of the 4-oxalocrotonate
tautomerase (4-OT) family can catalyze not only tautomerase and isomerase, but also dehalogenase reactions11,12. Pohl and colleagues have
used innovative mass spectrometry (MS)–based substrate screening
methods to determine that an archaeal enzyme originally annotated on
the basis of sequence analysis as a glucose-1-phosphate deoxythymidylyltransferase is actually a bifunctional acetyltransferase–N-acetylglucosamine-1-phosphate uridylyltransferase13. Notably, the MS-based
technologies described in this study should prove of general value for the
rapid and systematic biochemical characterization of new carbohydraterelated enzymes encoded by prokaryotic and eukaryotic genomes.
Similar functions from divergent sequences. Genomic enzymologists have also uncovered interesting cases where enzymes that share
no sequence or structural similarity perform similar functions. For
example, Begley and colleagues used comparative genomic analyses
to identify the first tryptophan-to-quinolinate pathway in bacteria14,
thereby dispelling the generally held belief that this process is unique
to eukaryotes. Before this discovery, prokaryotes were assumed to biosynthesize quinolinate exclusively from aspartate and dihydroxyacetone
131
REVIEW
a
E. coli
ycjG
+
dppA
ycjZ
ycjI
ycjY
dppB
dppC
mppA
=
dppD
dppE
ykfA
ykfB
ykfC
L-Ala-D-Glu
YcjG/YkfB
L-Ala-L-Glu
ykfD
B. subtilis
b
Substrate
Intermediate
O
O
Homologous
CO2-
O
N
CO2-
HN
O
N
H
H
HO
H
O
Mg2+
OOH
H
CH2OPO32-
O
O
H
Nonhomologous
HO
HO
Substrate
CH2OH
O
H
OH
HO
H
CH2OPO32-
OH
SCoA
O C O
CO2H
O
H
OH
HO
H
CH2OPO32HO
KGPDC
H
4-HBA-CoA
thioesterases
Arthrobacter
O
HN
N
Product
Product
Pseudomonas
Figure 3 Genomic enzymology. (a) The uncharacterized proteins YcjG and YkfB were determined to be L-Ala-D/L-Glu epimerases by integration of knowledge
of genomic context (the murein peptidoglycan metabolic pathway) and the mechanistic versatility of their parent enzyme class (enolase superfamily)9.
(b) OMPDC and KGPDC are homologous enzymes that catalyze mechanistically distinct reactions using different substrates10. OMPDC decarboxylates
orotidine 5′-monophosphate in a metal-independent manner, whereas KGPDC catalyzes the metal (Mg2+)-dependent decarboxylation of 3-keto-L-gulonate
6-phosphate. These intriguing differences have led to the proposal that these enzymes be considered members of a suprafamily, to designate that they share
significant sequence similarity but use distinct catalytic mechanisms. (c) The 4-hydroxybenzoate-CoA (HBA) thioesterase enzymes in the 4-chlorobenzoate
dehalogenation pathways of Arthrobacter and Pseudomonas share no sequence homology but perform the same reaction15.
phosphate. However, screening of genome archives identified clusters
of neighboring genes encoding homologs of key enzymes involved in
the eukaryotic tryptophan-to-quinolinate pathway in multiple bacterial
species. These bacterial enzymes were shown to produce quinolinate
from tryptophan by both biochemical and genetic methods. The predictive power of genomic context was once again exploited to identify an
unannotated gene with a metal-dependent hydrolase sequence as the
N-formylkynurenine formamidase component of the bacterial tryptophan-to-quinolinate pathway. These studies demonstrate that bacteria
can produce quinolinate, the universal de novo precursor to the pyridine
ring of NAD, through at least two distinct routes whose enzymatic constituents share no sequence, structural or mechanistic homology.
Dunaway-Mariano and colleagues have also provided an intriguing
example of sequence-unrelated enzymes that perform similar reactions. By comparing the 4-chlorobenzoate (4-CBA) dehalogenation
pathway operons from different bacteria, the authors readily identified
conserved sequence homologs responsible for the 4-CBA-coenzyme A
(4-CBA-CoA) ligase and 4-CBA-CoA dehalogenase activities in this
pathway15. Notably, however, the 4-hydroxybenzoate-CoA thioesterase
activity was found to be catalyzed by distinct sets of sequence-unrelated
enzymes (Fig. 3c). This divergence in protein sequences among 4-CBA
a
pathways from different bacteria suggests that they did not, as originally
suspected, evolve as a recent adaptation to exposure to industrially
produced 4-CBA, leading the authors to speculate that natural sources
of 4-CBA may exist.
Proteomic methods for the assignment of protein function
The comparative genomics methods described above are extremely powerful for prokaryotic organisms, where genes of related function are
often spatially linked in the genome. However, additional technologies
are needed to facilitate the global characterization of proteins encoded
by eukaryotic genomes. These technologies generally fit under the term
proteomics16,17. The daunting size and diversity of eukaryotic proteomes
has inspired efforts to profile specific classes of proteins on the basis
of shared functional properties18. Within this realm of ‘targeted proteomics’, chemical strategies are emerging as a powerful approach to
analyze protein function in complex biological systems19,20.
Profiling enzyme activities in proteomes. Conventional genomic and
proteomic methods comparatively quantify the expression levels of
transcripts21 and proteins22, respectively. From these abundance-based
measurements, changes in protein function are inferred. However, many
— 1 2 3 4 5 6
SDS-PAGE
RG/BG TAG
LC-MS
CE-MS
b
Rel. int.
© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
O
HN
OMPCD
c
OMPDC "suprafamily"
m/z
Inhibtor sensitivity
— 1 2 3 4
SDS-PAGE
132
Figure 4 Activity-based protein profiling (ABPP).
(a) General strategy for ABPP. Proteomes are
treated with chemical probes that label active
enzymes but not enzymes inhibited by intraor intermolecular regulators or those lacking
complementary binding sites. RG, reactive
group; BG, binding group; TAG, biotin and/or
fluorophore. Probe-labeled proteomes can be
analyzed through several platforms, including
gel24,27–29 or capillary32 electrophoresis and
LC-MS31,32. (b) Inhibitor discovery by ABPP.
The potency and selectivity of inhibitors can be
profiled in parallel by performing competitive
ABPP reactions in whole proteomes29,57,58.
VOLUME 1 NUMBER 3 AUGUST 2005 NATURE CHEMICAL BIOLOGY
REVIEW
Table 1 List of ABPP probes and their respective enzyme targets
Enzyme class
Electrophile/
Photo-cross-linker
References
Serine hydrolases
Electrophile
23, 24, 26
Cysteine proteases
Electrophile
37, 38, 40
Tag
Proteasome
Electrophile
45
Tag
Cathepsins
Electrophile
36
Ubiquitin hydrolases
Electrophile
39
Metalloprotease
Photo-cross-linker
41
Metalloprotease
Photo-cross-linker
42
Gamma-secretase
Photo-cross-linker
43
Serine/threonine
phosphatases
Electrophile
47
Tyrosine
phosphatases
Electrophile
46
Glycosidases
Electrophile
49
Kinases
Electrophile
48
Oxidoreductases
Enoyl-CoA hydratases
Glutathione-S-transferases (GSTs)
Electrophile
50–52
Hydroxypyruvate reductase
GSTs
Electrophile
35
Structure
O
EtO
Directed
n
P
Tag
F
Tag
O
H
N
X
R
N
H
O
HO2C
X = Halide or Acyloxy
O
O
S
N
H
© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
O
O
R
EtO
O
HO
N
H
R
Tag
N
H
Tag
H
N
N
H
O
O
H
N
OH O
O
H
N
Tag
Ubitquitin
N
H
O
N
H
O
O
O
HO
N
H
O
O
O
S
O
O
N
H
O
CF3
N
O
O
N
H
O
O
COOH
N
NH
O
O
O
R
O
HN
H
N
H
N
O
Tag
O
—O P
Tag
N
H
OH
HN
NH
N
O
OH
O
Br
H
N
OH
Tag
O
N3
HO
O
HO
F
F
Tag
O
O
MeO
O
O
H
O
O
O
Nondirected
R
R1
O
Cl
N
H
H
N
O
n
O
S
O
Tag
Tag
O
N
H
R2
CONH2
proteins, and in particular enzymes, are regulated by post-translational
mechanisms in vivo23, meaning that their expression level may not correlate with their activity. To address this important issue, a chemical
proteomic strategy has been introduced, referred to as activity-based
protein profiling (ABPP)24–26, that uses active site–directed probes to
read out the functional state of large numbers of enzymes directly in
whole proteomes (Fig. 4a). ABPP probes selectively label active enzymes
but not their inactive (for example, zymogen or inhibitor-bound)
forms27, facilitating the characterization of changes in enzyme activity
that occur in the absence of alterations in protein or transcript expression25,28,29. Additionally, because ABPP probes label enzymes on the
basis of shared catalytic properties rather than mere expression level,
they provide data sets enriched in low-abundance proteins that can
be read out in a range of formats, including gels28,29, microarrays30,
liquid chromatography31, and capillary electrophoresis32. These
features have enabled ABPP to be applied to a wide range of biological
samples, permitting, for example, the identification of new enzyme
activities elevated in aggressive cancer cells and tumors28,33,34, invasive
malaria parasites35 and obese livers36.
To date, we and others have generated ABPP probes for more than
20 enzyme classes, including members of all major families of proteases (serine24, cysteine37–41, metallo42,43, aspartyl44, proteasomal45,46),
phosphatases47,48, kinases49, glycosidases50 and glutathione S-transferases51 and a range of oxidoreductases31,51–53 (Table 1). These probes
have derived from a combination of two complementary approaches,
referred to as directed and nondirected ABPP54. In directed ABPP, probes
are designed to target a specific class of enzymes by the incorporation
of known affinity labels and/or binding groups into their structures.
Directed probes can achieve covalent modification of enzyme active
sites by either electrophilic labeling of complementary nucleophilic
residues24,37–41,45,46 or photo-cross-linking42–44. Because each directed
probe typically targets a large set of mechanistically related enzymes,
they have enabled the identification of unannotated members of enzyme
superfamilies that could not be classified by sequence analysis55,56. In
nondirected ABPP, combinatorial libraries of candidate probes are synthesized and screened against complex proteomes to identify specific
protein labeling events. Nondirected ABPP probes have used diverse
reactive groups, including sulfonate esters51,52 and α-chloroacetamides36
(Table 1), to target several mechanistically distinct enzyme classes.
Detailed analyses have provided strong evidence that, in most cases,
NATURE CHEMICAL BIOLOGY VOLUME 1 NUMBER 3 AUGUST 2005
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© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
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nondirected ABPP probes selectively label the active sites of their enzyme
targets31, indicating that they should provide an accurate assessment of
the functional state of these proteins in proteomes.
Although promiscuous probes are of great value for profiling many
enzyme activities in parallel, the assignment of protein function also
relies on the development of specific reagents to perturb individual
members of large enzyme classes. Toward this end, ABPP has proven
effective as a primary screen for the discovery of both reversible57 and
irreversible29,58 enzyme inhibitors (Fig. 4b). Inhibitor screening by
ABPP has several advantages over conventional substrate assays. First,
enzymes can be assayed in their native proteomic context without the
need for recombinant expression or purification. Second, inhibitors can
be developed for uncharacterized enzymes that lack known substrates57.
Finally, because inhibitors are evaluated against many enzymes in parallel, selective agents can be readily discriminated from nonspecific
compounds that target many enzymes. Inhibitor screening by ABPP
has culminated in the design of selective covalent probes for several
proteases, which have been used to test the function of these enzymes
in living systems and as in vivo imaging agents29,34.
One drawback of the original ABPP method is that probes were
encumbered with a large reporter tag, which can perturb their cellular uptake and distribution. Tag-free versions of ABPP were recently
introduced that exploit either the Huisgen’s azide-alkyne cycloaddition
reaction (click chemistry)59 or the Staudinger ligation46 to attach the
reporter group to activity-based probes after the covalent labeling of
protein targets. These advanced ABPP approaches have been used to
profile enzyme activities in vivo, resulting in the discovery of enzymes
that are selectively labeled in living cells, but not in vitro60.
Profiling post-translational modifications in proteomes. Chemical
strategies have also been developed to profile many types of post-translational modifications (PTMs) in cells and tissues. Protein phosphorylation events dynamically regulate the function of numerous intracellular
proteins and cell signaling pathways61. Phosphorylation sites are difficult to predict from sequence analysis alone; therefore, a major goal
of proteomics is to comprehensively map this PTM in both space and
time in cells and tissues. The Aebersold and Chait groups have described
methods to chemically tag phosphorylated peptides in proteomes for
their selective enrichment and characterization62,63. In the Aebersold
approach, phosphopeptides are converted to phosphoramidates,
which are then conjugated with cystamine and captured by reaction
with iodoacetyl groups immobilized on a solid support62 (Scheme 1a,
left). Chait and colleagues induced the base-catalyzed β-elimination
of phosphoserine and phosphothreonine residues to dehydroalanine,
which could then serve as a Michael acceptor for ethanedithiol and
subsequently be modified with a maleamide biotin tag63 (Scheme 1a,
right). Each of these approaches has distinct advantages and limitations, with the Aebersold method being applicable to all three types
of phosphorylation (serine, threonione, tyrosine) but requiring more
steps, whereas the Chait method is more efficient, but not applicable
to phosphotyrosine and susceptible to cross-reaction with O-linked
N-acetylglucosamine (O-GlcNAc)–modified peptides (see later discussion). Notably, a creative variant of this latter approach was recently
introduced by the Hathaway and Shokat groups, who showed that dehydroalanine peptides can be modified with cysteamine to create trypsin
cleavage sites for phosphorylation mapping by selective proteolysis64,65
(Scheme 1a).
Another dynamic PTM that is gaining considerable interest among
cell biologists is the O-GlcNAc modification of serine and threonine
residues66. O-GlcNAc has been shown to modulate a range of cellular
processes, including proteasomal degradation67 and gene silencing68.
134
Like phosphorylation, O-GlcNAc modifications are dynamic and
cannot be easily predicted from the analysis of protein sequences; thus,
technologies are needed for their direct identification in complex cell
and tissue preparations. Hart and colleagues showed that O-GlcNAc
modifications can be selectively eliminated over phosphorylation sites
under mild basic conditions, thus making them susceptible to analysis through the Michael acceptor chemical tagging method described
above69. More recently, the Bertozzi and Hsieh-Wilson groups have
described alternative strategies for tagging and enriching O-GlcNAc–
modified proteins from proteomes. Bertozzi and colleagues treated cells
with N-azidoacetylglucosamine (GlcNAz), resulting in the in situ delivery of a bio-orthogonal group (azide) to sites of O-GlcNAc modification in the proteome70 (Scheme 1b). GlcNAz-modified proteins could
then be derivatized through the Staudinger ligation with a reportertagged triarylphosphine ester for purification and characterization.
Notably, the Bertozzi group and others have exploited this metabolic
labeling method to profile additional PTMs, including mucin-type
O-linked glycosylation71 and lipid modifications such as farnesylation72,73.
Hsieh-Wilson and colleagues have taken a different approach and engineered a β-1,4-galactosyltransferase to append a ketone-containing
galactose selectively onto the C-4 hydroxyl of GlcNAc74 (Scheme 1c).
These ketone-tagged proteins are then reacted with an aminooxy biotin
reagent to allow their affinity purification by avidin chromatography75.
One advantage of this strategy is that it can be applied to any proteomic
sample, including those not readily amenable to metabolic engineering.
For example, 25 O-GlcNAc–modified proteins were identified from rat
forebrain, 23 of which were not previously known to be modified with
this carbohydrate75. Finally, a chemical labeling approach involving
oxidative cleavage and hydrazide reaction on solid phase has been developed to profile N-linked glycoproteins76. The inclusion of an isotopelabeling step permitted comparative quantification of glycoproteins in
different proteomic samples.
In summary, chemical tagging has emerged as a preferred strategy
for the selective enrichment and characterization of a range of PTMs in
proteomes. The molecular information garnered by these approaches
extends far beyond that which can be gleaned from standard genomics or proteomics. It should also be noted that the success of chemical
proteomic methods has been bolstered by tremendous advances in the
sensitivity and accuracy of modern protein MS techniques22. We anticipate that chemical proteomics will continue to play an integral part in
the inventorying of PTMs and assessment of the dynamic manner in
which these events regulate protein function in vivo.
Engineering specific enzyme-inhibitor pairs. Enzyme superfamilies
present an intriguing paradox for postgenomic researchers. On the
one hand, recognition from sequence, structural and/or biochemical
analyses that a protein belongs to a particular enzyme class restricts its
potential activities in vivo and therefore should, in principle, facilitate
functional characterization. However, the daunting size of enzyme
superfamilies complicates experimental efforts to assign unique
physiological activities to individual members. For example, classical
genetic approaches, such as targeted gene disruption, are susceptible
to compensatory changes by other superfamily members that mask
enzyme function. Likewise, inhibitors of enzymes from large classes
often lack the requisite selectivity for in vivo pharmacological application77. Recently, chemical biology strategies have been introduced that
integrate the complementary power of organic synthesis and protein
engineering to create tools for investigating the function of individual
members of enzyme superfamilies.
Protein kinases constitute the largest enzyme class in mammalian
proteomes and are key elements of most, if not all, signal transduction
VOLUME 1 NUMBER 3 AUGUST 2005 NATURE CHEMICAL BIOLOGY
REVIEW
+
NH3
a
H 2N
N
H
OPO3HH
N
1. Block
cysteine
O
OPO3HH
N
N
H
S
1. Base
O
H
N
N
H
HS
O
N H2
N
H
H
N
O
OH
O
2. Inactivate
cysteine
2. Trypsinize
1. HS
SH
O
2. Biotin
N
O
Proteome
1. Cap N and C termini
2. Regenerate phosphate
3. Cystamine derivative
O
Biotin
© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
O
Biotin
-O
BOCN
H
N
H
O
P N
H
O
H
N
N
S
N
S
S
O
SH
O
OH
N
H
O
1. Capture
2. Enrich
H2 N
OPO3HH
N
N
H
3. Release
1. Trypsinize
2. Enrich
S
O
OH
N
H
O
N
H
H
N
N
H
Enriched phosphopeptides
Protease
digestion
O
H
N
O
O
Enriched peptides
MS identification of phosphoproteins
and phosphorylation sites
b
OH
OH
O
HO
HO
O
OAc
O
AcO
AcO
Cells
OAc
OH
NH
N3
N3
TAG
TAG
O
O
NH
O
O
HO
HO
O
O
HO
HO
O
NH
O
N
H
PPh2
O
H
N
O
NH
O
OH
N3
TAG
O
HO
HO
O
TAG
O
O
NH
O
N3
SDS-PAGE; detect by western blot
c
TAG
OH
HO
HO
O
HO
OH
O
-O
OH
O
O
P
O
O
P
O -O O
O
OH
HO
HO
O
2.
H
HN
O
AcHN
O
N H
H
N
H
N
S
HN
O
OH
O
H
O
N H
H
O
1. Mutant GalT + HO
O
AcHN
OH
HO
NH
HO
AcHN
O
HO
HO
O
O
O
HO
O HO
O
N
N
H
O
O
AcHN
OH
TAG
H
N
S
O
O
N
H
O
TAG
NH2
Proteome
Protease
digestion
TAG
TAG
TAG
Mass
spectrometry
TAG
HO
Enrich
H
HN
TAG
O
N H
H
OH
O
H
N
S
O
O HO
O
N
N
H
OH
O
HO
O
AcHN
O
O
NH
Scheme 1 Chemical profiling of PTMs. (a) Chemical profiling of phosphoproteins. Two complementary methods for the chemical labeling, enrichment and
identification of phophorylation sites by LC-MS62,63. Also shown is a variant of one of these approaches, where the dehydroalanine intermediate is reacted
with cystamine to generate a lysine mimetic (orange) that enables phosphorylation site mapping by proteolysis64,65. (b) Chemical profiling of O-GlcNAc
modifications by metabolic labeling of cells70. Addition of Ac4GlcNAz to cells results in the incorporation of an azide into GlcNAc modification sites, which
are subsequently modified with a reporter TAG-phosphine (the Staudinger ligation) to enable affinity enrichment and characterization. (c) Chemical profiling
of O-GlcNAc modifications with the use of a mutant galactosyltransferase (GalT) enzyme that transfers a keto-sugar to O-GlcNAc sites in the proteome74,75.
The ketone is then reacted with a reporter TAG-oxime to enable affinity enrichment and characterization.
pathways that regulate cell physiology and pathology78. With more than
500 members encoded by the human genome, many still uncharacterized,
the ‘kinome’ constitutes a rich source for future biological discovery and
pharmaceutical intervention. To facilitate the functional characterization
of kinases, Shokat and colleagues have devised a general method to genetically engineer variants of these enzymes that interact with synthetic inhibitors and substrates not recognized by wild-type kinases79,80. Remarkably,
these ‘analog-sensitive’ (as) kinases can often be created by mutation of a
single active site residue, referred to as the ‘gatekeeper’ residue. Conversion
of this typically bulky residue to smaller amino acids, such as glycine, creates a ‘hole’ in the active site that can accept inhibitors and substrates (for
example, ATP analogs) bearing large substituents (‘bumps’) that sterically
clash with wild-type kinase binding pockets (Fig. 5a). This approach is
referred to as chemical genetics to signify that kinases have been genetically
NATURE CHEMICAL BIOLOGY VOLUME 1 NUMBER 3 AUGUST 2005
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a
a
NH2
NH2
N
N
N
Protein expression
N
N
O
N
N
N
Intein
S
NH2
© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
Wild type
as mutants
v-Src
c-Fyn
c-Abl
CDK2
CAMKII
v-Src-I338G
cFyn-T339G
c-Abl-T315A
CDK2-F80G
CAMKII-F89G
IC50(µM)
IC50(µM)
1
0.6
0.6
18
22
0.0015
0.0065
0.0070
0.015
0.097
28
1.0
3.4
29
24
0.0043
0.0032
0.12
0.0050
0.0080
b
Synthetic PTM
modified peptide
O
S
NH2
N
H
HS
O
N
H
Other
kinases
BCR-ABL
Imatinib
mesylate
KIT
BCR-ABL
Eliminates BCR-ABL+ KIT+
expressing cells
PKA
S—
O P
O
HO
ATP(γ)S
O
S
O
P
HO O
Alkylate
OH
hν
PDK1
kinase
KIT
H
N
O
Nonhydrolyzable
phosphotyrosine
PTM
b
Other
kinases
O
HO P O—
S N
Acyl Shift
Active
Inactive
c
Other
Kinases
BCR-ABL
as
KIT
Other
kinases
BCR-ABL
as
NaPP1
Insufficient to
eliminate BCR-ABL+ KIT+
expressing cells
KIT
Figure 5 Chemical genetic analysis of kinases. (a) Kinases are genetically
engineered to show sensitivity to inhibitors that are not active against wildtype kinases80. These as mutant kinases have been used to investigate the
mechanism of action of the cancer drug imatinib mesylate (b), showing that
it produces maximal cytotoxic effects by inhibiting multiple kinases (BCRABL and KIT)83.
engineered for sensitivity to specific chemical reagents.
Chemical genetics has been successfully implemented to test the roles
of many kinases in signaling events, both in culture and in vivo. For
example, chemical inhibition of an as mutant of the budding yeast kinase
Prk1p revealed a role for this protein in regulating actin patches involved
in endocytosis81. An as mutant of CamKIIα was introduced by transgenic methods selectively into the forebrains of mice, providing evidence
that the activity of this kinase is required during the first postlearning
week for memory consolidation82. Both of these studies highlight one
of the primary advantages of chemical genetic methods, namely, tight
temporal control over dynamic, kinase-regulated cellular and behavioral processes. An additional benefit was demonstrated by Witte and
colleagues, who showed that chemical genetics can be used to infer the
mechanism of action of kinase-directed inhibitors83. Specifically, selective inhibition of an as variant of the oncogenic kinase BCR-ABL did
not eliminate immature myeloid leukemic cells, which contrasts with the
cytotoxic effects of the BCR-ABL–directed inhibitor imatinib mesylate
(Gleevec) in these cells (Fig. 5b). These results suggest that the therapeutic efficacy of imatinib is caused by the inhibition of multiple kinases
in addition to BCR-ABL (for example, KIT, which is also expressed in
immature myeloid leukemic cells). Finally, as variants can also be used
in combination with ATP analogs to profile kinase substrate selectivity
in proteomic extracts, as exemplified by the studies of Morgan and colleagues on the cyclin-dependent kinase Cdk1 (ref. 84).
Given the impressive success of chemical genetic applications to
kinases, an obvious next question is whether this approach can be
generally extended to other enzyme superfamilies. Although studies
that speak to this issue are still relatively scarce, initial results with
136
O
O P O
O—
NO2
hν
RRLYRSLP
O
O P O—
O—
RRLYRSLP
Inhibition of
14-3-3 proteins
d
NO2
SH
Alkylate
S
CO2—
hν
Active
cofilin
Inactive
cofilin
Scheme 2 Introducing specific PTMs into proteins via chemical methods.
(a) The EPL method91, where proteins bearing specific phosphorylation
sites are semisynthesized for biophysical and cell biological studies. Inset
shows a nonhydrolyzable analog of phosphotyrosine used to characterize
the role of phosphorylation in SHP-2 (refs. 92,93). (b–d) Methods for
the creation of caged phosphorylation sites that can be unveiled by light.
(b) A caged phosphovariant of PKA generated with thiophosphorylation
chemistry95. (c) Caged phosphopeptides that bind 14-3-3 proteins
generated by solid-phase synthesis97,100. (d) A negative-regulatory
phosphorylation site in cofilin is regulated by conversion to either cysteine
(active) or a caged cysteine variant (inactive)101.
ATPases85, GTPases86,87, cyclophilins88 and phosphatases89 are
encouraging. Indeed, an as variant of myosin 1c has revealed a specific
role for this motor protein in controlling adapative responses in hair
cells of the inner ear85. Moreover, recent advances reported by Shokat
and colleagues to address kinase family members intolerant to original
gatekeeper mutations offer methodological and biophysical guidelines
that should assist future chemical genetics endeavors relating to this
enzyme family and others90.
Engineering specific post-translational modifications in proteins.
The historical view of a one-to-one relationship between gene and protein has been replaced by the provocative realization that numerous
protein products derive from a single gene2. These protein variants
arise by multiple mechanisms, including alternative splicing, proteolytic processing and PTM. Accordingly, as described above, a major
goal of proteomics is to inventory these epigenetic events to achieve a
more comprehensive understanding of the diversity of protein species
that exist in vivo. However, an equally if not more important problem
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a
c
O
O
OH
HO
OH O
O
OH
O
O
O
N
H
n
OH
OH
n
NAE
+
FFA
H2N
OH
Ethanolamine
OGlcGlc
H2 N
FAAH
OH O
PAL1
O
OH
OMe
Kaempferol
OH
HO
MeO
O
HO
Phe
GlcO
O
O
O
OH
OMe
Coumarins
© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
d
R
O
O
OH
mBCAT
O
+ HO
OH
NH2
O
Leu
α-KG
O
O
OH
O
α-KIC
N
OH
O
+ HO
SO3—
CYP +
cysteine
OH
n
NAT
Coniferyl
b
O
N
H
n
+
H2N
FFA
NH2
R
HO
S
CO2H
SUR1 +
glucose
N
Taurine
R
HO N
OH
NH2
SO3—
S
Glucose
PAPS +
sulfotransferase
CYP
Glu
O
R
OH
NH2
Primary
sulfur
metabolism
R
—
O3SO N
S
Glucose
Scheme 3 Identification of endogenous substrates of enzymes by global metabolite profiling. (a,b) Targeted metabolite profiling methods have been used
to explain (a) the role of PAL1 in the biosynthesis of phenylpropanoids (kaempferol, coniferyl, coumarins)105 and (b) a role for mBCAT in the generation
of branched chain α-keto acids106. Deletion of mBCAT leads to molecular and physiological phenotypes in mice that are reminiscent of human maple
syrup disease. (c) Discovery (untargeted) metabolite profiling identified a structurally new class of brain natural products, the NATs, that are regulated by
FAAH in vivo107. (d) Untargeted metabolite profiling, in combination with transcriptional profiling, identified the plant sulfotransferases involved in GLS
metabolism110.
is to determine the unique functions performed by different protein
isoforms. Indeed, the current (and rapidly expanding) catalog of PTMs
far exceeds the number of cases where the physiological significance
of these events has been deduced. The ability to create homogeneous
preparations of proteins bearing specific PTMs is essential for their biochemical and cell biological characterization. Multiple chemical biology
research programs have been initiated with this goal in mind.
Cole, Muir and colleagues have pioneered the development and
application of expressed protein ligation (EPL) methods for the semisynthesis of post-translationally modified proteins. These approaches
exploit the versatile native chemical ligation reaction, in which two
protein fragments, one containing a C-terminal thioester (generated
through fusion to an intein protein) and the other containing an Nterminal cysteine, react and rearrange through an S-to-N acyl shift
to form a native peptide bond91 (Scheme 2a). Significantly, protein
substrates for EPL can derive from recombinant or synthetic sources,
greatly expanding the range of chemical modifications that can be
site-specifically incorporated into proteins. Cole and colleagues have
used EPL to generate homogeneous preparations of singly and doubly phosphorylated forms of the tyrosine phosphatase SHP-2 (refs.
92,93). In these studies, a nonhydrolyzable phosphotyrosine mimetic
was incorporated into SHP-2, permitting dissection of the roles of specific phosphorylation events in regulating the biochemical and cellular
activity of this phosphatase. Muir and colleagues have exploited EPL
to decipher the contribution of individual phosphorylation events to
the oligomerization of Smad2 and its recognition as a substrate for
transforming growth factor β kinase94.
Chemical approaches have also been devised for the creation of caged
phosphopeptides or phosphoproteins, allowing exquisite spatial and
temporal control over their function in vitro and in living cells. Bayley
and colleagues have described the semi-synthesis of thiophosphorylated variants of protein kinase A (PKA), which is accomplished by
incubation of this protein with a kinase in the presence of ATP(γ)S95
(Scheme 2b). The thiophosphoryl group can then be selectively caged
on the basis of its enhanced nucleophilicity. Thiophosphoryl groups
have the advantage of being resistant to phosphatase cleavage and thus
show enhanced stability in living cells. One limitation of this approach,
however, is that enzyme-catalyzed thiophosphorylation reactions do
not yet constitute a generally applicable way to generate any phosphorylated protein.
Imperiali and colleagues have pioneered a complementary approach
that uses versatile 1-(2-nitrophenyl) ethyl–modified serine, threonine
and tyrosine building blocks for the synthesis of caged phosphoproteins
or phosphopeptides96,97 (Scheme 2c). These caged phosphoamino acids
can be incorporated into proteins or peptides through several mechanisms, including direct synthesis97, in vitro translation using nonsense
suppression methods98 and EPL99. Imperiali, Yaffe and colleagues have
applied caged phosphopeptides to disrupt the function of the 14-3-3
proteins in living cells100. Notably, with the use of a consensus phosphopeptide motif that interacts with all 14-3-3 proteins (Scheme 2c), this
chemical biology approach allowed the synchronous inactivation of an
entire family of adaptor proteins, a task that would have been difficult
to accomplish with traditional genetic methods.
Finally, Lawrence and colleagues have shown that the activity of
the actin-severing protein cofilin can be controlled by mutagenesis
of a serine phosphorylation site to either cysteine (active) or a caged
cysteine variant modified with α-bromo-(2-nitrophenyl) acetic acid
(inactive)101 (Scheme 2d). Photoactivation of the caged profilin in living cells revealed new roles for this protein in actin-based cytoskeletal
dynamics. This approach may prove generally useful for studying the
activity of proteins that are negatively regulated by phosphorylation.
It is clear from the examples provided here that chemical biology
approaches offer powerful methods and tools to test the functional
significance of specific PTMs. To date, most of these studies have
focused on protein phosphorylation, and it will be exciting to witness as
approaches such as EPL and caged amino acid derivatives are extended
to the analysis of additional PTMs. Toward this end, the ability to sitespecifically incorporate a range of amino acid derivatives into proteins
remains a paramount objective. Additionally, many proteins, such as
integral membrane proteins, are not easily produced in purified form
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© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
through either chemical synthesis or recombinant expression. Recent
advances by the Schultz and Muir groups that extend technologies such
as non-natural amino acid incorporation102,103 and EPL104 to in vivo
settings should expand the spectrum of post-translationally modified
proteins amenable to molecular and cellular analysis.
Metabolomic methods for the assignment of protein function
A protein’s function can be defined on many levels—molecular, cellular and physiological. Although these functions are all interrelated
(for example, an enzyme performs chemistry to generate a product
(molecular) that affects a cell (cellular), which in turn affects the higher
organism (physiological)), the technologies required for their experimental investigation are distinct. For example, genetic approaches such
as gene knockouts3 and RNAi4, as well as the proteomic methods delineated in the previous section, can be combined with cell- and organismbased phenotypic screens to characterize the cellular and physiological
functions of proteins. However, the endogenous biochemical activities
of proteins are less straightforward to address with these methods. For
enzymes, in particular, the identification of natural substrates often
remains an elusive goal. Recent advances in global metabolite profiling
offer powerful new ways to address this problem.
Enzymes regulate biological processes through the conversion of specific substrates to products. Therefore, the identification of an enzyme’s
full complement of physiological substrates is of paramount importance.
The systematic characterization of endogenous substrates of enzymes
requires the advancement of new technologies that can broadly profile
the molecular composition of cells and tissues. The field of metabolomics has emerged with this objective in mind. Metabolome researchers
are confronted with several technical challenges that, in many ways, surpass those of genomics and proteomics. First, as opposed to transcripts
and proteins, metabolites are not directly linked to the genetic code, but
are instead the products of the concerted action of complex enzymatic
networks. Similarly, metabolites are not linear polymers composed of a
defined set of monomers, but rather a remarkably diverse collection of
structures with differing chemical and physical properties. These distinguishing features have inspired the development of both targeted
and untargeted analytical methods for metabolite profiling that, when
coupled with genomic and proteomic technologies, provide a powerful
platform for forging direct connections among enzymes, substrates and
the higher-order physiological processes they regulate.
Assignment of enzyme function by targeted metabolite profiling.
Targeted metabolomic experiments have proven effective at characterizing the physiological relationship between a defined set of enzymes and
natural products. For example, by profiling the metabolic consequences
of disrupting distinct phenylalanine ammonia lyase (PAL) enzymes in
Arabidopsis thaliana, Rhode and colleagues succeeded in differentiating
their unique biochemical functions105. PAL enzymes had long been
recognized to participate in the biosynthesis of the phenylpropanoid
class of natural products (for example, lignins, flavonoids, coumarins);
however, which PAL enzymes performed these chemical transformations in vivo has remained unclear. Targeted liquid chromatographyultraviolet (LC-UV) spectroscopy revealed a primary role for PAL1 in
the biosynthesis of phenylpropanoids, with a secondary role for PAL2
(Scheme 3a). The authors also performed complementary genomic
studies on PAL gene mutants, leading to the identification of additional
metabolic pathways altered in these organisms. These results provided
the first global view of the extent to which individual PAL enzymes are
integrated into the larger metabolic networks of plant cells.
Targeted metabolite profiling has also been used as a screen to ‘molecularly phenotype’ mutant organisms originating from forward genetic
138
screens. Wu and colleagues profiled plasma amino acid and acylcarnitine
levels in ethylnitrosourea (ENU)-treated mice by direct-inject tandem
MS analysis, resulting in the discovery of a mutant mouse line with
highly elevated levels of branched-chain amino acids (BCAAs)106. These
mice also showed signs of wasting (reduced body weight and motility).
Similar phenotypes are observed in humans with defects in branchedchain α-keto acid dehydrogenase (BCKD), leading to maple syrup urine
disease. Surprisingly, however, the mutant mice did not show reduced
BCKD activity, but rather a loss in the function of the mitochondrial
branched-chain aminotransferase (mBCAT) enzyme, which converts
BCAAs to branched-chain α-keto acids (Scheme 3b), the natural substrates of BCKD. These results indicate that the disruption of one of
multiple enzymes in the BCAA biosynthetic pathway leads to a common
set of metabolic and physiological defects.
Assignment of enzyme function by untargeted metabolite profiling.
The targeted profiling methods described above provide a robust and
sensitive way to quantify known metabolites in complex biological
samples. However, these approaches are not suited for the discovery of
new metabolites and may therefore fail to report on the full spectrum
of substrates used by a given enzyme in vivo. To address this problem,
we have recently introduced a complementary liquid chromatographymass spectrometry (LC-MS)–based strategy for ‘untargeted’ metabolomics referred to as discovery metabolite profiling (DMP)107. In contrast
to targeted LC-MS, in which the levels of specific metabolites are measured by selected ion monitoring (SIM) in conjunction with isotopic
standards, DMP operates as a global, standard-free method, where the
relative levels of numerous metabolites are quantified in parallel by
the direct measurement of their mass ion intensities in a broad mass
scanning mode (Fig. 6).
DMP was applied to characterize the tissue metabolomes of wildtype mice and mice lacking the enzyme fatty acid amide hydrolase
[FAAH–/– mice], which degrades the N-acyl ethanolamine (NAE) class
of signaling lipids in vivo (Scheme 3c), including the endocannabinoid
anandamide108. Consistent with previous targeted LC-MS studies109,
DMP accurately measured an approximately ten-fold increase in the
central nervous system (CNS) levels of NAEs in FAAH–/– mice. The
detection of low-abundance NAEs such as anandamide indicated that
DMP maintained good sensitivity (estimated to be within one order
of magnitude of targeted methods). Interestingly, DMP also detected a
second class of CNS metabolites that were highly elevated in FAAH–/–
mice. These metabolites did not belong to any of the known classes
of endogenous lipids, suggesting that they constituted new natural
products. This premise was confirmed by subsequent analytical and
synthetic chemistry efforts, which identified the uncharacterized class
of FAAH-regulated lipids as conjugates of fatty acids and the amino
acid taurine [N-acyl taurines (NATs)] (Scheme 3c). The function of the
FAAH-NAT metabolic pathway in mammalian biology is under active
investigation. Finally, other major classes of known lipids, including
fatty acids, phospholipids, monoacylglycerols and ceramides, were not
altered in FAAH–/– mice, supporting a specific role for this enzyme in
the metabolism of endogenous amidated lipids. These results underscore the power of untargeted profiling methods such as DMP, which
can provide a more comprehensive portrait of the endogenous substrates of enzymes and, through doing so, illuminate unanticipated
connections between the proteome and metabolome.
Untargeted metabolite profiling has more recently been implemented, in combination with gene-expression profiling, to identify a
class of sulfotransferases involved in glucosinolate (GLS) metabolism
in plants110. The GLS metabolic pathway has been an active subject of
research for many years, and most of its participating enzymes had been
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a
340
m/z
360
80
60
40
20
m/z 347.5
Relative intensity
Mass ion intensity
100
Endogenous Deuterated
metabolite
standard
100
Endogenous
metabolite
Deuterated
standard
340
m/z
360
80
60
40
20
m/z 347.5
Retention time
Retention time
Wild type
Knockout
b
DMP (untargeted)
100
80
60
2
1
4
40
3
20
m/z 200–1200
Mass ion intensity
Mass ion intensity
© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
Mass ion intensity
Relative intensity
Targeted analysis
100
3
80
60
1
25
4
40
20
m/z 200–1200
Retention time
Retention time
Wild type
2
Ratio
KO/WT
Knockout
20
15
KO/WT 10
ratio
5
In summary, global metabolite profiling methods provide access to
a valuable portion of biomolecular space that is largely inaccessible to
genomic and proteomic methods. Significantly, much of the information provided by metabolomics, especially as relates to protein function
assignment, may be difficult if not impossible to procure by classical
biochemical methods. For example, the substrate selectivity of FAAH,
as determined in vitro with purified preparations of enzyme, was largely
unpredictive of the substrates used by this enzyme in vivo107. These
findings reinforce the importance of characterizing enzymes within the
context of the cells and tissues in which they are naturally expressed,
where potentially competitive and/or interdependent metabolic pathways are taken into account. Future efforts will probably focus on
expanding the portion of the metabolome that is addressable by DMP
methods. Lipids, which can be enriched by organic extraction, were an
obvious first target. The extension of DMP to other classes of metabolites will require the advent of methods for their selective enrichment.
Another important challenge will be to devise ways to accelerate the
determination of metabolite structures, possibly through the creation
of searchable databases that contain the chemical and physical features
of all known metabolites (for example, their mass fragmentation patterns, solvent solubility profiles, susceptibility to chemical modification). Solutions to these provocative problems, which may very well
come from the field of chemical biology, should greatly enhance our
understanding of how enzymes and their substrates interact to form
the higher-order metabolic and signaling networks that govern cell and
organismal behavior.
previously characterized, with the exception of the sulfotransferases.
Saito and colleagues monitored in an untargeted manner the timedependent changes in the metabolome and transcriptome induced by
sulfur deprivation using Fourier transform MS and DNA microarrays,
respectively. A collection of GLS metabolites and known biosynthetic
genes were found to show similar expression profiles. Interestingly,
within this collection of coregulated genes, the authors also found three
putative sulfotransferases. Recombinant expression and biochemical
analysis demonstrated that these proteins can indeed convert desulfoallyl GLS to sulfonated allyl GLS (Scheme 3d, red), confirming their
identification as GLS sulfotransferases. These findings indicate that the
integration of metabolomic and transcriptomic data can reveal functions for uncharacterized enzymes.
Summary and Future Directions
In this review, we have attempted to achieve two major goals. First, we
hope to have captured the remarkable power of integrated chemical
and biological approaches (dubbed ‘chemical biology’) for the determination of protein function. Whether it be the characterization of
new metabolic pathways in bacteria, or the identification of enzyme
activities and protein PTMs in mammalian proteomes, or the discovery of physiological substrates of enzymes, chemical biology efforts
are perhaps best defined by their motivation to unite methods and
tools from historically separate disciplines. This integrated approach,
where complementary techniques such as genetics, protein engineering, organic synthesis and analytical chemistry are brought to bear on a
single research problem, has allowed inquiries into the functional state
of proteins in highly complex biological samples, providing insights
into their molecular, cellular and physiological activities that extend far
beyond the information accessible to any individual discipline.
Our second objective was to illuminate the essentiality of genome
sequences to research endeavors that aim to describe protein function on
a systematic and global scale. Indeed, many of the technologies described
in this review are founded in classical experimental approaches, such as
operon analysis, affinity labeling and site-directed mutagenesis. However,
their conversion into global strategies for the characterization of protein
function was only possible with the availability of complete genome
sequences. For example, the sequencing of several hundred bacterial
genomes has fostered the emergence of new fields of research, such as
genomic enzymology, where biochemical, structural and comparative
genomic methods converge to reveal unexpected functional relationships among members of enzyme super- and suprafamilies. Likewise, the
molecular information procured by chemical proteomic investigations
of protein activity and PTM state would be virtually uninterpretable
without searchable databases of MS fragmentation patterns of all proteins encoded by a given genome. Finally, and perhaps most importantly, genome sequences provide a context in which to evaluate the
full complement of proteins from a given family to discern their unique
NATURE CHEMICAL BIOLOGY VOLUME 1 NUMBER 3 AUGUST 2005
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0
–5
–10
90 80 70
60 50 40 30
20
Retention
time
200
400
600
800 1,000
m/z
Global metabolite analysis
Figure 6 Comparison of targeted and untargeted LC-MS methods for
comparative metabolite analysis. (a) General scheme for targeted LC-MS
analysis, in which metabolites are detected by SIM (shown for a metabolite
with a mass of 347.5) and their levels are quantified by comparing mass
signals to those of isotopically distinct internal standards. (b) General
scheme for DMP107, an untargeted global LC-MS approach, in which
metabolites are detected in the broad mass scanning mode (for example,
200–1200 mass units) and their levels quantified by measurement of direct
mass ion intensities (that is, without the inclusion of internal standards).
Enzyme-regulated metabolites are identified by comparison of mass ion
intensity ratios between wild-type and knockout samples.
© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
REVIEW
and overlapping functions. In this regard, enzymes such as kinases and
hydrolases, for which there are several hundred representatives in the
human genome, stand as a humbling reminder of how much still remains
to be learned about even the most ‘familiar’ classes of proteins.
In looking to the future, we recognize several challenges that confront researchers interested in applying postgenomic methods to the
functional characterization of proteins. First, as the reader has probably
noticed, this review has placed a disproportionate emphasis on enzymes
over other types of proteins. We believe that this bias is rooted in several
factors. First, the function of enzymes, which generally equates with
catalytic activity, is, at least on first pass, simpler to define than for
many other proteins. Enzymes also possess well-defined active sites,
which harbor conserved catalytic residues that can be exploited to create class-wide approaches for experimental analysis (for example, sites
for comparative sequence alignment, affinity labeling or mutagenesis).
Nonetheless, we anticipate that many of the principles established for
the postgenomic examination of enzymes can be extended to other
types of proteins, such as receptors and ion channels, which also share
common molecular and structural features that should make them
vulnerable to unified strategies for functional characterization.
A more general problem relates to the somewhat arbitrary definition
of ‘protein function’ itself, which can vary depending on the mode of
scientific investigation. Indeed, it is becoming clear that many proteins in
both prokaryotic and eukaroytic organisms have been misannotated on
the basis of a cursory inspection of their sequences111. Although increasingly sophisticated sequence analysis programs such as Shotgun112,113
should help to rectify these errors, experimental approaches still stand
as the only satisfactory way to verify the actual activities performed by
proteins. Several of the methods described in this review use molecular
engineering to create specific variants of proteins for functional analysis.
Although these approaches offer the compelling advantage of isolating
one protein (or even one PTM state of a protein) for characterization in
complex biological settings, they generally involve heterologous expression systems and, thus, often do not address the function of proteins in
their native environment. In contrast, chemical profiling methods can be
applied to any biological system (even primary human biopsies) to assess
the functional state of natively expressed proteins. However, these profiling
technologies provide only an associative relationship between the activity or PTM state of proteins and a particular (patho)physiological event.
Follow-up studies are needed to deduce whether the identified proteins
actually contribute to the higher-order processes under investigation.
Given that molecular engineering and chemical profiling methods possess
complementary strengths and weaknesses, we suggest that future chemical biology efforts be aimed at fully developing these technologies such
that a complete spectrum of experimental approaches is available for the
functional characterization of all protein classes in the proteome. Before
the achievement of this ultimate goal, we can be satisfied with the fact that
chemistry, by providing unique reagents and methods for interrogating
the function of proteins in complex biological systems, promises to have
a prominent role in most, if not all, forms of postgenomic inquiry.
ACKNOWLEDGMENTS
The authors apologize to many wonderful colleagues whose work we were unable to
cite because of space limitations. We gratefully acknowledge the support of the US
National Institutes of Health (CA087660, DA017259, DA015197), the Helen L. Dorris
Child and Adolescent Neuropsychiatric Disorder Institute, and the Skaggs Institute for
Chemical Biology. A.S. is a Merck Fellow of the Life Sciences Research Foundation.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturechemicalbiology/
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C O R R I G E N D A A N D E R R AT U M
CORRIGENDUM: Assignment of protein function in the postgenomic era
Alan Saghatelian & Benjamin F Cravatt
Nat. Chem. Biol. 1, 130–142 (2005)
In Table 1, all reference numbers should be increased by 1, as shown.
© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology
Table 1 List of ABPP probes and their respective enzyme targets
Enzyme class
Electrophile/
Photo-cross-linker
Serine hydrolases
Electrophile
24,25,27
Cysteine proteases
Electrophile
38,39,41
Tag
Proteasome
Electrophile
46
Tag
Cathepsins
Electrophile
37
Ubiquitin hydrolases
Electrophile
40
Metalloprotease
Photo-cross-linker
42
Metalloprotease
Photo-cross-linker
43
γ-secretase
Photo-cross-linker
44
Serine/threonine
phosphatases
Electrophile
48
Tyrosine
phosphatases
Electrophile
47
Glycosidases
Electrophile
50
Kinases
Electrophile
49
Structure
O
EtO
Directed
References
n
P
Tag
F
Tag
O
H
N
X
O
HO2C
R
N
H
X = Halide or acyloxy
O
O
S
N
H
O
O
R
EtO
O
O
O
O
HO
N
H
R
N
H
OH O
Tag
H
N
N
H
O
O
H
N
O
H
N
Tag
Ubiquitin
N
H
O
N
H
O
O
O
S
HO
N
H
Tag
O
O
N
H
O
CF3
N
N
H
O
COOH
O
N
NH
O
O
O
R
O
O
O
P
HN
H
N
H
N
Tag
—O
Tag
N
H
OH
HN
NH
N
O
O
O
OH
O
Br
H
N
OH
Tag
O
N3
HO
O
HO
F
F
Tag
O
O
MeO
O
O
H
O
O
O
Nondirected
R
n
O
S
O
Tag
Oxidoreductases
Enoyl-CoA hydratases
Glutathione-S-transferases (GSTs)
Electrophile
R1
O
Cl
N
H
H
N
O
51–53
Tag
O
N
H
CONH2
Hydroxypyruvate reductase
GSTs
R2
NATURE CHEMICAL BIOLOGY VOLUME 1 NUMBER 4 SEPTEMBER 2005
Electrophile
36
233