Quantitative reactivity profiling predicts functional cysteines in proteomes.
ABSTRACT Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.
- Synlett 09/2014; 25(16):2239-2245. · 2.46 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: A very facile approach for the design and fabrication of a colorimetric sensor array, by using only a single indicator-receptor couple at various ratios and concentrations, is described for the first time. As a proof-of-concept application, discrimination and identification of the 20 natural amino acids has been successfully accomplished. Classification analyses demonstrate that the as-fabricated colorimetric sensor array has a high dimensionality and, consequently, has the capability to recognize the 20 natural amino acids. Moreover, the amino acids can be qualitatively and semi-quantitatively detected by combining classification analyses, recognition patterns and corresponding fitting curves. The strategy developed in the current study likely represents a “maximally” simplified approach for design and fabrication of colorimetric sensor arrays, and could be taken full advantage of among investigators in the sensing application field.RSC Advances 01/2014; 4(56):29581. · 3.71 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Biological thiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play important roles in maintaining the appropriate redox status of biological systems. The discrimination between them is of great importance because of their different biological roles. Herein, we present a new near-infrared (NIR) fluorescent sensor Cy-NO2 for selective detection of Cys over Hcy/GSH. The nitrothiophenol group is introduced to quench the fluorescence through photo-induced electron transfer (PET). The sensor undergoes displacement of nitrothiophenol with thiol to turn on the fluorescence. The amino groups of Cys/Hcy further replace the thiolate to form amino-substituted products, which exhibit dramatically different photophysical properties compared to the sulfur-substituted product from the reaction with GSH. By means of more rapid intramolecular displacement of sulfur with the amino group of Cys than Hcy, the discrimination of Cys is achieved. Moreover, Cy-NO2 was successfully applied for bioimaging Cys in living cells.RSC Advances 01/2014; 4(16):8360. · 3.71 Impact Factor
Quantitative reactivity profiling predicts
functional cysteines in proteomes
Eranthie Weerapana1,2*, Chu Wang1,2*, Gabriel M. Simon1,2, Florian Richter3,4, Sagar Khare3,5, Myles B. D. Dillon2,
Daniel A. Bachovchin1,2, Kerri Mowen2, David Baker3,4,5& Benjamin F. Cravatt1,2
Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse
biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered
their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic
reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature
among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis
and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized
function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and
is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the
basis for screeningand functional assignmentofcysteines incomputationally designed proteins, whereit discriminated
catalytically active from inactive cysteine hydrolase designs.
Large-scale scientific endeavours such as genome sequencing and
structural genomics are providing a wealth of new information on
organisms. Many of these proteins, however, remain partly or com-
pletely unannotated with respect to their biochemical activities1. New
methods are therefore needed to characterize protein function on a
global scale. Much effort is currently devoted to the characterization
of post-translational modification events because these covalent
adducts can have profound and dynamic effects on protein activity2.
Another frequently overlooked parameter that defines functional
‘hotspots’ in the proteome is amino acid side-chain reactivity, which
on local protein microenvironment. Methods to measure side-chain
been described, and as such, the reactive landscape of the proteome
remains largely unexplored.
Among the protein-coding amino acids, cysteine is unique owing
to its intrinsically high nucleophilicity and sensitivity to oxidative
modification. The pKaof the free cysteine thiol is between 8 and 9,
meaning that only slight perturbations in the local protein micro-
environment can result in ionized thiolate groups with enhanced
reactivity at physiological pH3. Diverse families of enzymes use
cysteine-dependent chemical transformations, including proteases,
oxidoreductases and acyltransferases4. In addition to its role in cata-
modification, including sulphenation (SOH), sulphination (SO2H),
nitrosylation (SNO), disulphide formation and glutathionylation,
which endow it with the ability to serve as a regulatory switch on
proteins that is responsive to the cellular redox state5.
or sites of post-translational modification, donot conform to a canon-
ical sequence motif, which complicates their systematic identification
and characterization.pKameasurementscan identifycysteineresidues
with heightened nucleophilicity (or ‘hyper-reactive’ cysteines6,7), but
this requires purified protein and detailed kinetic and mutagenic
experiments7,8that cannot be performed on a proteome-wide scale.
Additional methods have been introduced to computationally predict
and qualitatively inventory electrophile-modified cysteines in pro-
teomes15–18. Some of these studies have provided suggestive evidence
that nucleophilic cysteines may possess a variety of important func-
tions14–18, although the non-quantitative methods used in each case
ship. We adopted a different strategy to globally characterize cysteine
functionality in proteomes based on quantitative reactivity profiling
with isotopically labelled, small-molecule electrophiles.
Quantifying cysteine reactivity in proteomes
Our approach, termed isoTOP-ABPP (isotopic tandem orthogonal
proteolysis–activity-based protein profiling), has four features to
enable quantitative analysis of native cysteine reactivity (Fig. 1a): (1)
an electrophilic iodoacetamide (IA) probe, to label cysteine residuesin
proteins, that also has (2) an alkyne handle for ‘click chemistry’ con-
jugation of probe-labelled proteins19to (3) an azide-functionalized
TEV-proteaserecognition peptide containing a biotingroup for strep-
of IA-labelled peptides across multiple proteomes (Supplementary
Fig. 1). After tandem on-bead proteolytic digestions with trypsin
and TEV protease15,20, probe-labelled peptides attached to isotopic
tags are released and analysed by liquid-chromatography-high-
resolution MS to identify IA-modified cysteines and quantify their
extent of labelling based on MS2 and MS1 profiles, respectively. An
isoTOP-ABPP ratio, R, is generated for each identified cysteine that
reflects the difference in signal intensity between light and heavy tag-
amounts of a mouse liver proteome (13, 23, 43) with the IA probe
*These authors contributed equally to this work.
USA.3Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.4Interdisciplinary Program in Biomolecular Structure and Design, University of Washington, Seattle,
Washington 98195, USA.5Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA.
0 0 M O N T H 2 0 1 0 | V O L 0 0 0 | N A T U R E | 1
Macmillan Publishers Limited. All rights reserved
followed by click chemistry conjugation with either the heavy or light
cysteines closely matched the expected proteome ratios (R1:1<1,
ative MS/MS profile of an IA-labelled peptide from our proteomic
experiments is provided in Supplementary Fig. 3.
In contrast to traditional cysteine-alkylating protocols for proteo-
mics that use millimolar concentrations of IA to stoichiometrically
modify all cysteines in denatured proteins21, we proposed that, by
applying low (micromolar) concentrations of the IA probe to native
proteomes, differences in the extent of alkylation would reflect differ-
ences in cysteine reactivity, rather than abundance. This hypothesis
concentrations of IA probe, where hyper-reactive cysteines would be
expected to label to completion at low probe concentrations (generat-
ing isoTOP-ABPP ratios with R[high]:[low]<1) and less reactive
labelling (generating isoTOP-ABPP ratios with R[high]:[low]?1)
isoTOP-ABPP experiments with the soluble proteome of the human
breast cancer cell line MCF7 using pair-wise IA-probe concentrations
of 10:10mM, 20:10mM, 50:10mM and 100:10mM (light:heavy). More
than 800 probe-labelled cysteines were identified on 522 proteins, the
vast majority of which exhibited escalating isoTOP-ABPP ratios
(Fig. 1b) expected for reactions that did not reach completion over
the tested probe concentration range. In contrast, a small subset of
cysteines (,10%) showed nearly identical ratios at all probe concen-
trations tested (R1:1<R2:1<R5:1<R10:1<1, Fig. 1b, shaded blue
box). An expanded analysis of multiple human cancer line (Sup-
plementary Fig. 5 and Supplementary Table 1) and mouse tissue
(SupplementaryFig. 6 andSupplementary Table 2) proteomestreated
with low (10mM) and high (100 mM) IA-probe concentrations
revealed consistent isoTOP-ABPP ratios for individual cysteine resi-
dues, indicating that the propensity of a cysteine to display high IA
reactivity is an intrinsic property of the residue (and presumably its
specific to a particular cell or tissue. Additionally, isoTOP-ABPP
ratios showed no correlation with either protein abundance or pep-
tide ion intensity (Supplementary Fig. 7), indicating that they were
independent of potential MS-based ionization sources for saturation.
Finally,weconfirmed thatsimilarisoTOP-ABPP ratios were obtained
for cysteines in reactions where time rather than the concentration of
probe was varied (Supplementary Fig. 8 and Supplementary Table 3),
(hyper-reactivity), rather than saturable binding interactions (see
Hyper-reactivity predicts cysteine functionality
We next sought to assess the functional ramifications of the special
subset of cysteines that showed hyper-reactivity in isoTOP-ABPP
experiments. We first noted that multiple sites of IA-probe labelling
on the same protein often showed markedly different isoTOP-ABPP
on four cysteine residues, three of which showed high ratios (C90,
C192 and C237 had ratios of R10:155.6, 7, and 5.4, respectively),
whereas the fourth (C32) showed a low ratio of R10:150.9 (Fig. 2a).
Interestingly, C32 is the active-site nucleophile of GSTO1 (ref. 22).
Acetyl-CoA acetyltransferase-1 (ACAT1) was also labelled on four
ratios of R10:158.8, 8.2 and 4, respectively), whereas the fourth, the
active site nucleophile C126(ref. 23), yielded alowratioof R10:151.1
might be a good predictor of cysteine functionality in proteins. To
examine this premise more systematically, we queried the Universal
Protein Resource (UniProt) database to retrieve functional annota-
tions for the 1,082 cysteine residues labelled by the IA probe. This
analysis revealed that the most hyper-reactive cysteines were remark-
ably enriched in functional residues, with 35% of the cysteines with
R10:1,2 being annotated as active-site nucleophiles or redox-active
disulphides compared to 0.2% for all cysteine residues in the UniProt
of hyper-reactive cysteines identified several that have been ascribed
functional properties in the literature despite lacking annotation in
cysteine C108 (R10:151.0) was identified in the uncharacterized
protein D15Wsu75e. This protein and its orthologues are predicted
K. DYEFM W NPHLGYI LTC*PSNLGTGLR. A
R10:1 = 1.18
K. M VM TVFAC*LM GK. G
R10:1 = 10.13
Light TEV tag
Heavy TEV tag
2. Avidin enrichment
3. Trypsin digestion
4. TEV digestion
Figure 1 | A quantitative approach to globally profile cysteine reactivity in
proteomes. a, isoTOP-ABPP involves proteome labelling, click-chemistry-
based incorporation of isotopically labelled cleavable tags, and sequential on-
bead protease digestions to provide probe-labelled peptides for MS analysis.
The IA probe is shown in the inset. LC-MS/MS, liquid-chromatography-MS/
MS. b, Measured isoTOP-ABPP ratios for peptides from MCF7 cells labelled
with four pairwise IA probe concentrations (10:10mM, 20:10mM, 50:10mM,
100:10mM). The blue box highlights peptides with low isoTOP-ABPP ratios
peptides in blue and red, respectively, and green lines depicting peak
heavy-labelled peptides with green lines representing predicted values.
Sequences are shown for tryptic peptides containing IA-probe-labelled
cysteines (marked by asterisks) in CKB and LCP1. RT, retention time.
Additional chromatographs from isoTOP-ABPP experiments are in
Supplementary Table 7.
2 | N A T U R E | V O L 0 0 0 | 0 0 M O N T H 2 0 1 0
Macmillan Publishers Limited. All rights reserved
to be cysteine proteases based on conservation of a prototypical Cys-
His catalytic dyad24. Interestingly, C108 corresponds to the putative
cysteine nucleophile of this catalytic motif and a recent crystal struc-
ture confirms the proximity of C108 to a conserved histidine (H38)
(Supplementary Fig. 12). Thus, quantitative reactivity profiling sup-
ports structural predictions that D15Wsu75e is a functional cysteine
Hyper-reactive cysteines also corresponded to sites for post-
translational modification. For instance, C101 (R10:151.92) in the
protein arginine methyltransferase PRMT1 has been identified as a
site of modification by the endogenous oxidative product 4-hydroxy-
2-nonenal (HNE)25. This cysteine, although nonessential for catalytic
function, is an active site residue that makes direct contact with the
S-adenosylmethionine cofactor26(Fig. 3a). Interestingly, we found that
HNE inhibited both the IA-labelling (Fig. 3b) and catalytic activity
(Fig. 3c) of wild-type PRMT1. A C101A mutant of PRMT1 showed
substantially reduced IA-labelling (Fig. 3b) and HNE sensitivity
stress pathways through selective HNE modification of its hyper-
reactive, active-site C101 residue. Additional hyper-reactive cysteines
represented sites for glutathionylation27(CLIC1 (C24), CLIC3 (C25)
and CLIC4 (C35); R10:152.02, 1.07 and 1.45, respectively) and nitro-
sylation28(RTN3; C42, R10:150.78). These data, taken together, indi-
but also of ‘non-catalytic’, active-site cysteines, as well as those that
undergo various forms of oxidative modification.
Function of the hyper-reactive cysteine in FAM96B
cysteines, wereasonedthatcritical activitiesmightbeinferredforsuch
displaying low isoTOP-ABPP ratios uncovered the highly conserved
C93 (R10:151.15) in the uncharacterized protein FAM96B (Sup-
Saccharomyces cerevisiae, which shows 52% identity with human
FAM96B, including conservation of C93 (the corresponding residue
in YHR122W is C161). The gene encoding YHR122W is essential for
but not the C161A mutant of YHR122W could rescue a yeast strain in
which the YHR122W gene was conditionally suppressed (Fig. 4a and
R10:1 < 2.0
2.0 < R10:1 < 5.0
R10:1 > 5.0
Annotated active site + redox-active disulphide
Annotated structural disulphide and others
No functional annotation
Per cent functionally annotated
Annotated active-site nucleophile
or redox-active disulphide
Figure 2 | Hyper-reactive cysteines are highly enriched in functional
residues. a, Chromatographs from an isoTOP-ABPP experiment using
100:10mM IA probe are shown for peptides from GSTO1 (top) and ACAT1
(bottom). The cysteine nucleophiles (asterisks) show low ratios (R10:1<1),
whereas other cysteines show high ratios (R10:1$4). b, Pie charts illustrating
the percentage of functionally annotated cysteines for three isoTOP-ABPP
ratio ranges, including an average derived from all cysteines in the UniProt
database. c, Correlation of isoTOP-ABPP ratios with functional annotations
from the UniProt database where active-site nucleophiles or redox-active
(window of 50) of functional residues is shown as a dashed blue line,
demonstrating a profound enrichment within R10:1,2.0. Data are from
experiments in three human cancer cell lines (MCF7, MDA-MB-231 and
HNE (μM)HNE (μM)
Figure 3 | Functional characterization of the hyper-reactive cysteines in
PRMT1. a, Crystal structure of rat PRMT126(green, PDB accession code
1ORI) showing the hyper-reactive cysteine C101 in contact with an
mutant of human PRMT1 were labelled with the IA probe, followed by click
chemistry to incorporate a fluorescent rhodamine tag. In-gel fluorescence
demonstratesrobust labelling of the wild-typebutnotC101AmutantPRMT1,
and shows that IA-probe labelling of wild-type PRMT1 is inhibited by HNE
(upper panel). Lower panel shows Coomassie blue staining for treated protein
samples. c, Catalytic activity of purified wild-type, but not C101A mutant
from3H-S-adenosylmethionine (SAM) to a histone 4 substrate.
0 0 M O N T H 2 0 1 0 | V O L 0 0 0 | N A T U R E | 3
Macmillan Publishers Limited. All rights reserved
the FAM96B family.
We also observed that expression of the C161A mutant of
YHR122W caused a severe growth defect in non-suppressive media
tary Fig. 14). This result indicates that the YHR122W protein may
engage in protein complexes that are sequestered by the C161A
mutant, thereby disrupting the activity of the wild-type protein.
Consistent with this premise, queries of the Saccharomyces genome
databank (SGD) revealed that YHR122W has been found in several
large-scale protein interaction studies to bind to proteins involved in
cytosolic iron-sulphur (FeS) cluster assembly, namely Nar1 and Cia1
(ref. 30; Fig. 4b). We found that the activity of the FeS-client protein
isopropylmalate isomerase (Leu1)31was markedly reduced in
support a role for the YHR122W/FAM96B protein in FeS-protein
biogenesis. We also note that reactive cysteines seem to be a common
human orthologues of Nar1, Met18 and Cfd1 (NARF, MMS19 and
NUBP2, respectively) (R10:150.91, 2.2 and 2.9 respectively) (Sup-
FeS clusters to client proteins32.
Predicting functional cysteines in designed proteins
The marked correlation between cysteine hyper-reactivity and func-
tionality observed in native proteomes led us to ask whether this rela-
IA labelling of twelve proteins that were computationally designed to
act as cysteine hydrolases. These proteins originated from structurally
distinct scaffolds and were all designed to contain cysteine-histidine
dyads within an active site cavity (see Supplementary Methods for
more details). Two of the designed proteins, ECH13 and ECH19,
showed significant hydrolytic activity using a fluorogenic ester sub-
strate, whereas the other ten designs were inactive (Fig. 5a and
Supplementary Fig. 15a).
We first evaluated IA labelling of protein designs using a clickable,
fluorescent reporter tag and SDS–polyacrylamide gel electrophoresis
(SDS–PAGE) analysis, where similar amounts of each protein were
tested inahomogeneous backgroundproteome representing amix of
Escherichia coli and human (MCF7 cell line) proteins. The two active
compared to inactive designs (Fig. 5a), and, in bothcases, mutation of
the active-site cysteine to alanine abolished labelling (Fig. 5b) and
containing all twelve protein designs, diluted them into a background
human cell proteome, and analysed the mixture by isoTOP-ABPP.
Notably, both ECH13 and ECH19 showed isoTOP-ABPP ratios that
were equivalent to the most hyper-reactive cysteines in human and
E. coli proteomes (R10:150.92 and 1.27, respectively), whereas the
remaining inactive protein designs all showed higher ratios ranging
from 1.88–6.11 (Fig. 5c and Supplementary Fig. 15b, c). These data
thus reveal a strong correlation between cysteine hyper-reactivity and
hydrolytic activity across a diverse panel of protein designs and
designate heightened cysteine nucleophilicity as a key feature of suc-
cessful cysteine hydrolase designs.
Here, we have described a quantitative method to profile the intrinsic
reactivityofcysteine residues in native proteomes. Measurementofthe
enzymologists to assess the nucleophilicity of cysteine residues in indi-
extended to quantitative, proteome-wide surveys of cysteine reactivity
in complex biological systems. A key advantage of isoTOP-ABPP over
more traditional proteomic methods that target cysteine-containing
biotinylated reagents, which have shown an impaired ability to label
permeability, also afford the opportunity to perform cysteine reactivity
profiling in living systems. In pilot experiments, we have found that a
tively targets probe-accessible cysteines in native proteins. In this way,
structural cysteines engaged in disulphide bonds or buried within the
fraction of cysteines that are profoundly enriched in functionality (the
Enzyme activity (%)
Figure 4 | Functional characterization of YHR122W/FAM96B.
a, Expression of wild type and a C161A mutant of YHR122W in a yeast strain
with a doxycycline (dox)-repressable YHR122W gene demonstrated a
dominant-negative phenotype on induction of the C161A mutant expression
(2dox/1gal, middle panel) and rescue of viability by expression of wild type,
but not the C161A mutant of YHR122W (1dox/1gal, right panel). b, The
cytosolic FeS cluster assembly pathway contains multiple proteins with hyper-
this network based on protein–protein interaction studies (see http://
www.yeastgenome.org/). This panel was adapted from ref. 30. c, Doxycycline
treatmentof theYHR122W-repressableyeast strainsignificantly decreased the
activity of the cytosolic FeS enzyme Leu141, and this activity is rescued by
overexpression of wild-type YHR122W. These treatments had no effect on the
activity of the non-FeS enzyme alcohol dehydrogenase (ADH). Error bars
represent standard deviation, n53. ***P,0.001, Student’s t-test.
4 | N A T U R E | V O L 0 0 0 | 0 0 M O N T H 2 0 1 0
Macmillan Publishers Limited. All rights reserved
IA probe labelled 1,082 out of a total of 8,910 cysteines present on the
890 human proteins detected in this study). Projecting forward, it is
possible that, by varying the nature of the electrophile, isoTOP-ABPP
probes can be created that profile the reactivity of different subsets of
cysteines, as well as other amino acids in proteomes, such as serine,
threonine, tyrosine and glutamate/aspartate, which have also been
shown to react with small-molecule probes16,18,33–35.
We discovered that hyper-reactivity can predict cysteine function
in both native and designed proteins. The fact that hyper-reactivity
was strongly correlated with catalytic activity in de novo designed
cysteine hydrolases is interesting from the principles of both enzyme
engineering and assay development, as it indicates that heightened
for novel cysteine-dependent enzymes. We show that these screens
can be performed directly in complex proteomes using either gel or
MS (isoTOP-ABPP) detection platforms, thus offering aversatile and
relatively high-throughput way to evaluate many protein designs in
parallel. The isoTOP-ABPP platform has the additional advantage of
reading out the relative cysteine reactivity of designs independent of
cysteines for comparison. isoTOP-ABPP might also offer a com-
plementary way to perform cysteine reactivity/accessibility experi-
ments that monitor protein stability and ligand interactions36,37.
beyond nucleophilic catalysis to include other enzymatic activities
fication. Quantitative reactivity profiling thus distinguishes itself as a
complementary and perhaps more inclusive strategy to survey cysteine
function compared to previous computational9and experimental11–14,17
methods that focus on specific cysteine-based activities or modification
events. Considering further that hyper-reactive cysteines corresponded
we speculate that cysteine nucleophilicity is a property that may have
been selected for during evolution to offer points of protein control by
oxidative stress pathways. Determining how the reactivity of cysteine
residues is honed will require further investigation, but we anticipate
that quantitative proteomic data, when integrated with the output of
not conform to any obvious consensus sequence motifs, many of these
17). This finding is consistent with literature reports ascribing a role
for a-helix dipoles in the stabilization of cysteine thiolate anions38.
Finally, it is important to stress that some functional cysteines may
be inherently reactive, but inaccessible to our IA probe for steric
reasons. Other cysteine-reactive electrophilic probes16,17may prove
more suitable for such cysteine residues. Also, hyper-reactivity is not
necessarily a defining feature for all functional cysteines. Some
enzymes with catalytic cysteines may, for instance, show reduced
reactivity until they bind their physiological substrates or may rely
tion. This may be the case with the E1-activating and E2-conjugating
enzymes, which recognize a specific class of ubiquitinated substrates
and possess active-site cysteines that showed only moderate levels of
electrophile reactivity (Supplementary Fig. 18). Other cysteines may
do indicate, however, that those cysteines that are hyper-reactive in
proteomes probably perform important catalytic and/or regulatory
functions for their parent proteins. The large number of newly dis-
covered residues that fall into this category foretell a broad role for
hyper-reactive cysteines in mammalian biology.
biotin tags were synthesized as previously described20,39.
Sample preparation, mass spectrometry and data analysis. For concentration-
dependent experiments, proteome samples in PBS were probe labelled with the
desired probe concentration for 1h. Click chemistry was performed with either
the light or heavy variants of the azide-TEV-biotin tags and the samples were
digestion. The resulting TEV digests were analysed by Multidimensional Protein
Identification Technology (MudPIT) on an LTQ-Orbitrap instrument. The
resulting tandem MS data were searched using the SEQUEST algorithm40using
EA28EA27EA24EA22 ECH192ayh11ajk3FR26 ECH13ECH12ECH10 ECH06
Activity – – – + – – –– – – –+
isoTOP-ABPP ratio (R)
0100200 300400500 600
R = 0.92 1.271.88 2.053.91 4.705.115.53 6.11
Figure 5 | Quantitative reactivity profiling predicts functional cysteines in
designed proteins. a, In-gel fluorescence demonstrates robust IA labelling of
two active cysteine hydrolases, ECH13 and ECH19, relative to inactive designs
velocities in the presence versus the absence of purified enzymes were
71.6466.94 and 104.15610.78, respectively (see Supplementary Fig. 15a for
substrate hydrolysis assay). Other designs showed no measurable hydrolysis
activity over background (0.7660.058 nmols21). Asterisks designate
Coomassie blue signals for protein designs (lower panel). b, IA labelling is
observed for ECH13 and ECH19, but not their active-site cysteine mutants
C45A and C161A, respectively. c, Catalytic cysteines in ECH13 and ECH19
show low isoTOP-ABPP ratios (red) compared with other designs (blue).
experiment (bottom panel), in the same order as shown in the top panel.
0 0 M O N T H 2 0 1 0 | V O L 0 0 0 | N A T U R E | 5
Macmillan Publishers Limited. All rights reserved
a concatenated target/decoy variant of the human, mouse and E. coli protein
sequence databases. Quantification of light:heavy ratios (isoTOP-ABPP ratios,
R) was performed using in-house software. Detailed information on sample
preparation, mass spectrometry methods and data analysis is presented in
DNA encoding wild-type YHR122W was subcloned into the pESC_Leu vector
procedure (Stratagene). These constructs were introduced into a yeast Tet pro-
moter Hughes (yTHC) strain harbouring a conditional (doxycycline-dependent)
disruption in the YHR122W gene (Open Biosystems). Growth of these trans-
formed cell lines on 6gal/6dox media was monitored for 3 days. These cell lines
were also used to monitor Leu1 and alcohol dehydrogenase (ADH) activity.
Detailed information on the protocols used to subclone, transform and monitor
the growth of the yeast strains and measure enzyme activity is available in
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 21 May; accepted 3 September 2010.
Published online 21 November 2010.
1.Eisenberg, D., Marcotte, E. M., Xenarios, I. & Yeates, T. O. Protein function in the
post-genomic era. Nature 405, 823–826 (2000).
Zhao, Y. & Jensen, O. N. Modification-specific proteomics: strategies for
Proteomics 9, 4632–4641 (2009).
Bulaj, G., Kortemme, T. & Goldenberg, D. P. Ionization reactivity relationships for
cysteine thiols in polypeptides. Biochemistry 37, 8965–8972 (1998).
Biophys. Res. Commun. 300, 1–4 (2003).
cysteine oxidation. Curr. Opin. Chem. Biol. 12, 746–754 (2008).
Voss, A. A., Lango, J., Ernst-Russell, M., Morin, D. & Pessah, I. N. Identification of
Biol. Chem. 279, 34514–34520 (2004).
Lewis, C. T., Seyer, J. M. & Carlson, G. M. Cysteine 288: an essential hyperreactive
thiol of cytosolic phosphoenolpyruvate carboxykinase (GTP). J. Biol. Chem. 264,
Knowles, J. R. Intrinsic pKa-values of functional-groups in enzymes: improper
deductions from pH-dependence of steady-state parameters. CRC Crit. Rev.
Biochem. 4, 165–173 (1976).
Fomenko, D. E., Xing, W., Adair, B. M., Thomas, D. J. & Gladyshev, V. N. High-
throughput identification ofcatalytic redox-activecysteine residues. Science 315,
10. Sethuraman, M. et al. Isotope-coded affinity tag (ICAT) approach to redox
proteomics: identification and quantitation of oxidant-sensitive cysteine thiols in
complex protein mixtures. J. Proteome Res. 3, 1228–1233 (2004).
11. Baty, J. W., Hampton, M. B. & Winterbourn, C. C. Proteomic detection of hydrogen
12. Salsbury, F. R. Jr, Knutson, S. T.,Poole, L. B. & Fetrow, J. S. Functional site profiling
andelectrostatic analysis ofcysteinesmodifiabletocysteine sulfenic acid.Protein
Sci. 17, 299–312 (2008).
13. Leonard, S. E., Reddie, K. G. & Carroll, K. S. Mining the thiol proteome for sulfenic
acid modifications reveals new targets for oxidation in cells. ACS Chem. Biol. 4,
14. Kim,J.-R.,Yoon,H.W.,Kwon,K.-S.,Lee,S.-R.& Rhee,S.G.Identificationofproteins
containing cysteine residues that are sensitive to oxidation by hydrogen peroxide
at neutral pH. Anal. Biochem. 283, 214–221 (2000).
15. Speers, A. E. & Cravatt, B. F. A tandem orthogonal proteolysis strategy for high-
content chemical proteomics. J. Am. Chem. Soc. 127, 10018–10019 (2005).
16. Weerapana, E., Simon, G. M. & Cravatt, B. F. Disparate proteome reactivity profiles
of carbon electrophiles. Nature Chem. Biol. 4, 405–407 (2008).
17. Dennehy,M.K.,Richards,K.A.,Wernke,G.R.,Shyr,Y.& Liebler,D.C.Cytosolicand
nuclear protein targets of thiol-reactive electrophiles. Chem. Res. Toxicol. 19,
18. Shin, N.-Y., Liu, Q., Stamer, S. L. & Liebler, D. C. Protein targets of reactive
19. Speers, A. E., Adam, G. C. & Cravatt, B. F. Activity-based protein profiling in vivo
using a copper(I)-catalyzed azide-alkyne [3 1 2] cycloaddition. J. Am. Chem. Soc.
125, 4686–4687 (2003).
20. Weerapana, E., Speers, A. E. & Cravatt, B. F. Tandem orthogonal proteolysis-
of probe modification in proteomes. Nature Protocols 2, 1414–1425 (2007).
21. Shiio, Y. & Aebersold, R. Quantitative proteome analysis using isotope-coded
affinity tags and mass spectrometry. Nature Protocols 1, 139–145 (2006).
22. Board, P. G. et al. Identification, characterization, and crystal structure of the
omega class glutathione transferases. J. Biol. Chem. 275, 24798–24806 (2000).
23. Thompson, S. et al. Mechanistic studies on b-ketoacyl thiolase from Zoogloea
ramigera: identification of the active-site nucleophile as Cys89, its mutation to
Ser89, and kinetic and thermodynamic characterization of wild-type and mutant
enzymes. Biochemistry 28, 5735–5742 (1989).
24. Iyer, L. M., Koonin, E. V. & Aravind, L. Novel predicted peptidases with a potential
role in the ubiquitin signaling pathway. Cell Cycle 3, 1440–1450 (2004).
25. Codreanu, S. G., Zhang, B., Sobecki, S. M., Billheimer, D. D. & Liebler, D. C. Global
analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal. Mol.
Cell. Proteomics 8, 670–680 (2009).
26. Zhang, X. & Cheng, X. Structure of the predominant protein arginine
methyltransferase PRMT1 and analysis of Its binding to substrate peptides.
Structure 11, 509–520 (2003).
channel CLIC1 (NCC27) at 1.4-A˚resolution. J. Biol. Chem. 276, 44993–45000
28. Hao, G., Derakhshan, B., Shi, L., Campagne, F. & Gross, S. S. SNOSID, a proteomic
method for identification of cysteine S-nitrosylation sites in complex protein
mixtures. Proc. Natl Acad. Sci. USA 103, 1012–1017 (2006).
29. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome.
Nature 418, 387–391 (2002).
30. Lill, R. Function and biogenesis of iron sulphur proteins. Nature 460, 831–838
31. Pierik, A. J., Netz, D. J. & Lill, R. Analysis of iron–sulfur protein maturation in
eukaryotes. Nature Protocols 4, 753–766 (2009).
32. Netz, D. J. A., Pierik, A. J., Stumpfig, M., Muhlenhoff, U. & Lill, R. The Cfd1–Nbp35
complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol.
Nature Chem. Biol. 3, 278–286 (2007).
33. Okerberg, E. S. et al. High-resolution functional proteomics by active-site peptide
profiling. Proc. Natl Acad. Sci. USA 102, 4996–5001 (2005).
34. Nazif, T. & Bogyo, M. Global analysis of proteasomal substrate specificity using
positional-scanning libraries of covalent inhibitors. Proc. Natl Acad. Sci. USA 98,
35. Chen, G.et al.Reactivity offunctionalgroups onthe protein surface:development
36. Silverman, J. A. & Harbury, P. B. Rapid mapping of protein structure, interactions,
protein stability and function by quantitative cysteine reactivity. Proc. Natl Acad.
Sci. USA 107, 4908–4913 (2010).
38. Kortemme, T. & Creighton, T. E. Ionisation of cysteine residues at the termini of
model a-helical peptides. Relevance to unusual thiol pKavalues in proteins of the
thioredoxin family. J. Mol. Biol. 253, 799–812 (1995).
39. Macpherson,L.J.etal.Noxiouscompounds activateTRPA1ionchannels through
covalent modification of cysteines. Nature 445, 541–545 (2007).
40. Eng, J. K., Mccormack, A. L. & Yates, J. R. An approach to correlate tandem mass-
spectral dataofpeptides withamino-acid-sequences inaprotein database.J.Am.
Soc. Mass Spectrom. 5, 976–989 (1994).
are essential for biogenesis of cytosolic Fe/S proteins. EMBO J. 18, 3981–3989
Supplementary Information is linked to the online version of the paper at
Acknowledgements We would like to thank T. Bartfai, I. Wilson and members of the
B.F.C. laboratory for comments and critical reading of the manuscript, T. Ji for
experimental assistanceandJ.Gallaherfor expression ofdesignedproteins.Thiswork
was supported by the National Institutes of Health (CA087660, MH084512), a Pfizer
Postdoctoral Fellowship (E.W.), a Koshland Graduate Fellowship in Enzyme
Biochemistry (G.M.S.), a National Science Foundation predoctoral fellowship (D.A.B.)
and the Skaggs Institute for Chemical Biology.
Author Contributions B.F.C., E.W. and C.W. conceived the project and E.W. and C.W.
computational data analyses. S.K., F.R. and D.B. performed computational design of
cysteine hydrolases and measured activity using a fluorogenic assay. D.A.B. purified
PRMT1 and M.B.D.D. andK.M.performedPRMT1 activityassays. B.F.C.,E.W., C.W.and
G.M.S. analysed data and wrote the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to B.F.C. (email@example.com).
6 | N A T U R E | V O L 0 0 0 | 0 0 M O N T H 2 0 1 0
Macmillan Publishers Limited. All rights reserved
All compounds and reagents were purchased from Novabiochem, Sigma or
Fisher, except where noted.
and immediately flash frozen in liquid nitrogen. The tissues were then Dounce
homogenized in 13 PBS, pH 7.4. Centrifugation at 100,000g (45min) provided
stored at 280uC till use.
Preparation of human cancer cell line proteomes. MDA-MB-231 cells were
free incubator. Jurkat cells and MCF7 cells were grown in RPMI-1640 supple-
mented with 10% fetal bovine serum at 37uC with 5% CO2. For in vitro labelling
experiments, cells were grown to 100% confluency, washed three times with PBS
and scraped in cold PBS. Cell pellets were isolated by centrifugation at 1,400g for
MDA-MB-231 and MCF7 cells, the cells were grown to 90% confluency, the
media was removed and replaced with fresh media containing 10mM IA probe.
The cells were incubated at 37uC for 1h and harvested as detailed above. The
harvested cell pellets were lysed by sonication and fractionated by centrifugation
(100,000g, 45min) to yield soluble and membrane proteomes. The proteomes
were diluted to 2mgml21and stored at 280uC until use.
Protein labelling and click chemistry. Proteome samples were diluted to a 2mg
labelling reactions were incubated at room temperature (25uC) for 1h. Click
chemistry was performed by the addition of 150mM of either the light TEV tag
or heavy TEV tag (15ml of a 5mM stock), 1mM tris(2-carboxyethyl)phosphine
1:4) and 1mM CuSO4(503 stock in water). Samples were allowed to react at
room temperature for 1h. After the click chemistry step, the light- and heavy-
labelled samples were mixed together and centrifuged (5,900g, 4min, 4uC) to
pellet the precipitated proteins. The pellets were washed twice in cold MeOH,
after which the pellet was solubilized in PBS containing 1.2% SDS via sonication
and heating (5min, 80uC).
For time course experiments, proteome samples were labelled with 100mM of
IA probe (using 5ml of a 10mM stock in DMSO). After 6min of probe labelling,
5 column (GE Healthcare) to remove excess, unreacted probe. After 60min of
performed as described earlier.
(Pierce) for 3h at room temperature. The beads were washed with 10ml 0.2% SDS/
(1,300g, 2 min) between washes.
On-bead trypsin and TEV digestion. The washed beads described earlier were
and placed in a 65uC heat block for 15min. Twenty millimolar iodoacetamide
Following reduction and alkylation, the beads were pelleted by centrifugation
(1,300g, 2min) and resuspended in 200ml of 2M urea/PBS, 1mM CaCl2
(1003 stock in H2O), and trypsin (2mg). The digestion was allowed to proceed
overnight at 37uC. The digest was separated from the beads using a Micro Bio-
Spin column and the beads were then washed with 3 3 500ml PBS, 3 3 500ml
H2O, and 1 3 150ml of TEV digest buffer. The washed beads were then resus-
pendedin 150mlof TEVdigestbufferwith AcTEVProtease(Invitrogen,5ml) for
12h at 29uC. The eluted peptides were separated from the beads using a Micro
was added to the sample, which was stored at 220uC until MS analysis.
Liquid-chromatography-mass-spectrometry (LC-MS) analysis. LC-MS/MS
analysis was performed on an LTQ-Orbitrap mass spectrometer (ThermoFisher)
TEV digests were pressure loaded onto a 250mm fused silica desalting column
packed with 4cm of Aqua C18 reverse phase resin (Phenomenex). The peptides
were then eluted onto a biphasic column (100mm fused silica with a 5mm tip,
packed with 10cm C18 and 3cm Partisphere strong cation exchange resin (SCX,
Whatman) using a gradient 5–100% buffer B in buffer A (buffer A:95% water, 5%
acetonitrile, 0.1% formic acid; buffer B: 20% water, 80% acetonitrile, 0.1% formic
acid). The peptideswerethenelutedfromthe SCX onto the C18resinandintothe
mass spectrometer using four salt steps as previously described15,20. The flow rate
through the column was set to ,0.25mlmin21and the spray voltage was set to
2.75kV. One full MS scan (FTMS) (400–1,800MW) was followed by 18 data
dependent scans (ITMS) of the nth most intense ions with dynamic exclusion
Peptide identification. The tandem MS data were searched using the SEQUEST
algorithm40using a concatenated target/decoy variant of the human and mouse
International Protein Index databases. A static modification of 157.02146 on
cysteine was specified to account for iodoacetamide alkylation and differential
modifications of 1464.28596(light probe modification)and 1470.29977(heavy
probe modification) were specified on cysteine to account for probe modifica-
tions with the either light or heavy variants of the IA-probe-TEV adduct.
SEQUEST output files were filtered using DTASelect 2.042. Reported peptides
were required to be fully tryptic and contain the desired probe modification and
discriminant analyses were performed to achieve a peptide false-positive rate
below 5%. The actual false-positive rate was assessed at this stage according to
established guidelines43and found to be ,3.5%. Additional assessments of the
false-positive rate were performed following the application of additional filters
(described later) resulting in a final false-positive rate below 0.05%.
Ratio quantification. Quantification of light/heavy ratios (isoTOP-ABPP ratios,
that utilizes routines from the open-source XCMS package44for MS data analysis
to read in raw chromatographic data in the mzXML format45. Each experiment
consisted of two LC/LC-MS/MS runs: light:heavy 10mM:10mM, and light:heavy
and filtered with DTASelect as described earlier. Because the mass spectrometer
was configured for data-dependant fragmentation, peptides are not always iden-
tified in every run. As such, peptides were identified in either 1) only the
10mM:10mM run, 2) only the 100mM:10mM run, or 3) both runs. In the case of
peptides that were sequenced in both runs, identification of the corresponding
the case ofprobe-modified peptides that were sequencedinone, but not the other
run, an algorithm was developed to identify the corresponding peak in the run
‘reference’ peptide is used to position a retention time window (610min) across
the run lacking a peptide identification. Extracted ion chromatograms (6 10
p.p.m.) of the target peptide m/z with both ‘light’ and ‘heavy’ modifications are
pairs of light:heavy MS1 peaks, and for each candidate pair calculates the ratio of
integrated peak area between the light and heavy peaks. Several filters are used to
spectrum. This comparison generates an ‘envelope correlation score’ (Env) that
also enables confirmation of the monoisotopic mass and charge state of each
candidate peak. Peak pairs that have poor co-elution scores, or that have the
incorrect monoisotopic mass or charge, or whose isotopic envelopes are not well
correlated with the predicted envelope are eliminated from consideration. After
applicationofthese filters, in the rare case that multiplecandidates stillexist,then
filters results in a single candidate peak pair and the ratio for this peak pair is
recorded for the peptide in the corresponding run. In this way, each experiment
Following application of these filters, the false-positive rate was reassessed, and
found to be less than 0.05% in all cases.
After ratios for unique peptide entries are calculated for each experiment,
overlapping peptides with the same labelled cysteine (for example, same local
sequence around the labelled cysteines but different charge states, MudPIT seg-
ment numbers, or tryptic termini) are grouped together, and the median ratio
fromeachgroupis reportedas thefinalratio (R). Allof thesevaluescanbe found
in Supplementary Tables 1, 2 and 3 and representative chromatographs can be
seen in Supplementary Table 7. Raw result files of peptide identification using
SEQUEST can be found in Supplementary Table 9.
custom perl-scripts were developed to query the UniProtKB/Swiss-Prot Protein
Knowledgebase release 57.4 (current as of 16 June 2009). Sequence annotationin
the (Features) section of the relevant UniProt entry was mined and any annota-
tion corresponding to the labelled residue was collected. This functional annota-
tion in its entirety can be found in Supplementary Tables 4 and 5.
Recombinant PRMT1 protein expression and purification. Full-length cDNA
encoding human PRMT1 in pOTB7 was purchased from Open BioSystems and
was grown in LB media containing 75mgl21carbenicillin with shaking at 37uC
Macmillan Publishers Limited. All rights reserved
to an OD600nmof 0.5. The cells were then induced with 1mM isopropyl-b-D-
thiogalactoside(IPTG)andharvested4h laterby centrifugation. Cellswerelysed
supplemented with 1mgml21lysozyme and 1mgml21DNase I. The lysate was
then sonicated and centrifuged at 10,000g for 10min. Talon cobalt affinity resin
(Clontech; 400ml of slurry per gram of cell paste) was added to the supernatant,
and the mixture was rotated at 25uC for 30min. Beads were collected by cent-
rifugationat 700g for 3min, washedtwicewithTris buffer,and appliedto a 1-cm
column. Thecolumnwas washedtwice withTris buffer(10ml per 400ml ofresin
the addition of 100mM imidazole (2ml per 400ml of resin). Imidazole was
removed by passage over a Sephadex G-25M column (GE Healthcare), and the
eluate was concentrated using an Amicon centrifugal filter device (Millipore).
Protein concentration was determined using the Bio-Rad DC protein assay kit.
These conditions yielded PRMT1 at approximately 0.5mgl21of culture. A
ing mutant protein was expressed identically and isolated with a similar yield.
In-gel fluorescence characterization of PRMT1. Thirteen micrograms of
recombinant PRMT1 (wild type or C101A mutant) in 50ml PBS buffer was
pre-incubated with 0, 25 or 50mM HNE (Calbiochem, 50mM stock in ethanol)
for 1h at room temperature and was then labelled with 100nM of the IA probe
(5mM stock in DMSO) and the reactions incubated for 1h at room temperature.
Click chemistry was performed with 20mM rhodamine-azide, 1mM TCEP,
100mM TBTA ligand and 1mM CuSO4. The reaction was allowed to proceed
at room temperature for 1h before quenching with 50ml of 23 SDS–PAGE
loading buffer (reducing). Quenched reactions were separated by SDS–PAGE
(30ml of sample/lane) and visualized in-gel using a Hitachi FMBio IIe flatbed
laser-induced fluorescence scanner (MiraiBio).
PRMT1 in vitro methylation assays. Five-hundred nanograms of recombinant
human PRMT1 (wild type or C101A mutant) was pre-incubated with HNE
(Calbiochem) for 30min and methylation activity was monitored after addition
of 1mg of recombinant histone 4 (M2504S; NEB) and SAM (2mCi) in methyla-
tion buffer (20mM Tris, pH 8.0, 200mM NaCl, 0.4mM EDTA). Reactions were
incubated for 90min at 30uC and stopped with SDS sample buffer. SDS–PAGE
gels were fixed with 10% acetic acid/10% methanol v/v, washed, and incubated
with Amplify reagent (Amersham) before exposing at 280uC.
Complementation of S. cerevisiae YHR122W deletion mutant. A cDNA
encoding YHR122W was purchased as a full-length expressed sequence tag
(Open Biosystems). The construct for subcloning into the yeast epitope tagging
from the corresponding cDNA using the following primers: sense primer,
59-GAAGCGGCCGCAATGTCTGAGTTTTTGAATGA-39; antisense primer,
digested pESC-Leu vector and sequenced. The YHR122W(C161A) mutant was
sequenced and found to contain only the desired mutation.
Constructs containing wild-type and C161A mutant YHR122W were intro-
duced into the yTHC strain YSC1180-7428770 (Open Biosystems) using the
reagents provided in the Yeastmaker Yeast Transformation System 2 (Clontech).
The yeast was grown in synthetic dextrose minimal medium (2Leu) and spot
assays were performed in either synthetic dextrose minimal medium (2Leu) or
synthetic galactose minimal medium (2Leu) 1 agar plates 6 50mgml21doxycy-
cline. The plates were cultured at 30uC for 3 days.
Isopropylmalate isomerase (Leu1) assay. Yeast strains harbouring either an
empty vector or wild-type YHR122W (see earlier section) were cultured in syn-
synthetic galactose minimal medium (2Leu) 6 50mgml21doxycycline for 12h.
Yeast were lysed and Leu1 semi-purified by ammonium sulphate precipitation
(40–70%). The activity assays were performed using DL-threo-3-isopropylmalic
acid as the substrate and product formation was measured by monitoring absor-
bance at 235nm for 10min31.
ADH assay. Yeast cell lysates in 0.1M sodium pyrophosphate buffer (pH 9.2,
1.5ml) were treated with 2M ethanol (0.5ml) and 0.025M NAD (1.0ml) and
ADH activity was measured by absorbance increase at 340 nm for 3 min47.
the Rosetta computational enzyme design methodology48to search a set of
protein scaffolds for constellations of backbones capable of supporting an idea-
lized transition state for ester hydrolysis derived from the geometries and
mechanisms of natural cysteine hydrolases49. The idealized active-site models
feature a nucleophilic cysteine, a general base/acid histidine and at least one
of residues surrounding the putative active sites was optimized using the Rosetta
designalgorithmto maximizetransitionstatestabilization50. Asetof12designed
eachdesigned protein,synthetic genes wereobtained and proteinexpressionand
purification was performed in E. coli as previously described50. Activity was
measured with the substrate by following the initial (,5% substrate conversion)
concentration of 20mM and substrate concentration of 100mM were used in
25mM HEPES buffer, 150mM NaCl, 1mM TCEP, pH 7.5. The background rate
was measured under identical conditions but without the protein. Kunkel muta-
genesis was used for creating point mutations in the active-site residues. A
detailed description of the design and characterization of the cysteine hydrolases
in Supplementary Information.
In-gel fluorescence and isoTOP-ABPP characterization of designed proteins.
of the IA probe (5mM stock in DMSO) and the reactions incubated for 1h at
room temperature. Click chemistry, SDS–PAGE separation and in-gel fluor-
escence visualization were performed as described in previous sections.
For isoTOP-ABPP studies, 10ul of each of the E.coli lysates (2mg protein/ml)
was brought to 1ml by the addition of 2mgml21of MCF7 soluble proteome.
Time-dependent and concentration-dependent labelling with the IA probe, click
chemistry, on-bead trypsin and TEV digestions, LC-MS runs and MS data ana-
lysis were performed as described in previous sections.
42. Tabb, D. L., McDonald, W. H. & Yates, J. R. III. DTASelect and Contrast: tools for
assembling and comparing protein identifications from shotgun proteomics. J.
Proteome Res. 1, 21–26 (2002).
43. Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in
large-scale protein identifications by mass spectrometry. Nature Methods 4,
44. Collins, S. R. et al. Toward a comprehensive atlas of the physical interactome of
Saccharomyces cerevisiae. Mol. Cell. Proteomics 6, 439–450 (2007).
45. Pedrioli, P. G. A. et al. A common open representation of mass spectrometry data
and its application to proteomics research. Nature Biotechnol. 22, 1459–1466
mass spectrometry-based proteomics. Nature Methods 5, 319–322 (2008).
47. Vallee,B. L.& Hoch, F. L. Zinc, a component ofyeastalcoholdehydrogenase.Proc.
Natl Acad. Sci. USA 41, 327–338 (1955).
enzyme design. Protein Sci. 15, 2785–2794 (2006).
J. Am. Chem. Soc. 129, 13633–13645 (2007).
50. Jiang, L. et al. De novo computational design of retro-aldol enzymes. Science 319,
Macmillan Publishers Limited. All rights reserved