R ES E A RC H

◥ (Fig. 1A). As such, we sought to pursue redox re-

RESEARCH ARTICLE activity as an alternative strategy for methionine
ligation and now report a method, termed redox-
activated chemical tagging (ReACT), that en-
CHEMICAL BIOLOGY ables chemoselective methionine bioconjugation
(Fig. 1A) in proteins and proteomes.

Redox-based reagents Development of ReACT for chemoselective

methionine bioconjugation
for chemoselective Inspired by observations of facile autoxidation
of methionine residues to methionine sulfoxides

methionine bioconjugation during mass spectrometry analyses, we reasoned

that an oxidative sulfur imidation reaction (29)
might serve as an attractive starting point for the
Shixian Lin,1* Xiaoyu Yang,1,8* Shang Jia,1 Amy M. Weeks,6 Michael Hornsby,6 ReACT strategy, owing to the flexibility of intro-
Peter S. Lee,6 Rita V. Nichiporuk,4 Anthony T. Iavarone,4 James A. Wells,6,7 ducing various functionalities on the nitrogen
F. Dean Toste,1,5† Christopher J. Chang1,2,3,5† pendant (Fig. 1A and fig. S1). We initiated our
study by screening a variety of sulfur imidation
Cysteine can be specifically functionalized by a myriad of acid-base conjugation strategies for reactions with methionine derivative S1 as a
applications ranging from probing protein function to antibody-drug conjugates and model substrate in 1:1 CD3OD/D2O solvent using
proteomics. In contrast, selective ligation to the other sulfur-containing amino acid, methionine, proton nuclear magnetic resonance (1H NMR)
has been precluded by its intrinsically weaker nucleophilicity. Here, we report a strategy for analysis of substrate conversion and reaction se-
chemoselective methionine bioconjugation through redox reactivity, using oxaziridine-based lectivity between the desired N-transfer product

Downloaded from [Link] on October 01, 2023

reagents to achieve highly selective, rapid, and robust methionine labeling under a range of (NTP, sulfimide) and unwanted O-transfer prod-
biocompatible reaction conditions. We highlight the broad utility of this conjugation method to uct (OTP, sulfoxide) (Fig. 1B). Surveys of various
enable precise addition of payloads to proteins, synthesis of antibody-drug conjugates, and transition metal–catalyzed sulfur imidation re-
identification of hyperreactive methionine residues in whole proteomes. actions were unfruitful, resulting in either no
conversion or formation of sulfoxide as the only

S
product (fig. S1). However, a strain-driven sulfur
ulfur occupies a privileged place in biology In contrast to the substantial body of literature imidation of methionine using oxaziridine 1 (Ox1)
owing to its versatile and unique chemistry on cysteine bioconjugation, analogous methods as the sulfur imidation reagent afforded 95% con-
(1). Although cysteine and methionine are for methionine labeling under physiological con- version of S1 within 2.5 min without additional
the only two sulfur-containing proteinogenic ditions remain largely underdeveloped. Despite catalyst with a NTP:OTP ratio of 5:1 (Fig. 1B).
amino acids, the sulfur center plays a diverse a number of compelling motivations for this pur- On the basis of previous reports that oxaziridines
array of critical roles spanning catalysis to metal suit, previous methods have generally employed substituted with an electron-withdrawing group
binding to redox regulation and other posttrans- highly electrophilic reagents to convert methio- (EWG) favored formation of the OTP (30), a car-
lational modifications (2–4). In this context, se- nine to sulfonium salts (21, 22) at low pH. Methi- bamate substituted with a weak EWG was ini-
lective protein conjugation methods based on onine is among the most hydrophobic and the tially examined (Fig. 1C). From this starting point,
cysteine modification have enabled a broad range second rarest amino acid in vertebrates, and taken altering the linkage of the probe from carbamate
of fundamental and applied advances (5, 6), from together with the fact that the majority of methi- to a weaker electron withdrawing urea (Ox2) re-
probes of protein function (4, 7–9) to synthesis onine residues are buried within interior protein sulted in enhanced selectivity (NTP:OTP = 12:1)
of covalent small-molecule inhibitors (10, 11) and cores (1), surface-accessible methionines are lim- with comparable conversion. Further attempts
antibody-drug conjugates (12) to activity- and ited and offer a potentially valuable handle for to tune electronic effects by substitution of the
reactivity-based protein profiling for functional highly selective protein modification using natu- benzylic hydrogen of Ox2 with an electron with-
cysteine identification (7, 13, 14) and inhibitor de- rally occurring amino acid side chains. In addi- drawing CF3 group (Ox3) resulted in much lower
velopment (15) (Fig. 1A). Cysteine bioconjugation tion, posttranslational modifications of methionine, selectivity (NTP:OTP = 2:1) and reaction conver-
strategies typically exploit the intrinsically high including by oxidation and/or metal binding (2, 23), sion (58%), likely as a result of increased steric
nucleophilicity of the thiol/thiolate side chain, in- are emerging as critical nodes in signaling path- hindrance. We observed a marked improvement
cluding elegant methods based on electrophilic ways that control function at the cell and orga- in NTP:OTP selectivity from 6:1 to 18:1 by increas-
warheads such as maleimides and alkyl and aryl nism level. For example, reversible oxidation of ing the percentage of water in the solvent medium
halides (16, 17), transition metal–mediated bio- specific methionine residues within actin can con- from 0 to 95% (Fig. 1B). In accord with previously
conjugation (18), and cysteine-to-dehydroalanine trol its assembly and disassembly to serve as a posited hypotheses (30, 31), this improvement
conversion (16, 19, 20). navigational signal (24, 25), and the antioxidant likely results from increased stabilization of the
function of methionine sulfoxide reductase has transition state leading to intermediate A, which
been linked to regulation of life span (26). In ad- should be improved by solvation and hydrogen
1
Department of Chemistry, University of California, Berkeley, CA, dition, recent work suggests that methionine oxi- bonding to the developing alkoxy anion (Fig. 1C).
USA. 2Department of Molecular and Cell Biology, University of
California, Berkeley, CA, USA. 3Howard Hughes Medical Institute,
dation can also increase binding interactions with Together, these data presage the utility of this
University of California, Berkeley, CA, USA. 4California Institute aromatic residues within proteins (27). ligation reaction in biological environments.
for Quantitative Biosciences, University of California, A major chemical challenge in developing a We next evaluated the reactivity of oxaziridine
Berkeley, CA, USA. 5Chemical Sciences Division, Lawrence selective methionine modification reaction under probes with other biologically relevant amino acid
Berkeley National Laboratory, Berkeley, CA, USA.
6
Department of Pharmaceutical Chemistry, University of
pH-neutral physiological conditions is its rela- competitors. In all cases, we did not observe any
California, San Francisco, CA, USA. 7Department of Cellular tively weak nucleophilicity, which precludes the conjugation products with any of the other ami-
and Molecular Pharmacology, University of California, San traditional approach of identifying an appropri- no acids tested; only methionine gave a ligated
Francisco, CA, USA. 8School of Physical Science and ate methionine-specific electrophilic partner for product with the ReACT reagent. Cysteine as well
Technology, ShanghaiTech University, Shanghai, China.
*These authors contributed equally to this work. †Corresponding
its acid-base bioconjugation in the presence of as selenocysteine were oxidized to their cystine
author. Email: chrischang@[Link] (C.J.C.); fdtoste@ competing, more nucleophilic amino acids such forms, with no NTP formation observed (fig. S2),
[Link] (F.D.T.) as cysteine, lysine, tyrosine, or serine (16, 22, 28) attesting to the high selectivity of the ReACT

Lin et al., Science 355, 597–602 (2017) 10 February 2017 1 of 6

R ES E A RC H | R E S EA R C H A R T I C LE

Fig. 1. The ReACT strategy for chemoselective

methionine bioconjugation. (A) (Left) Acid-base
conjugation strategies for cysteine-based protein
functionalization. Iodoacetamide (IAA) and malei-
mide reagents are representative electrophiles for
selective cysteine bioconjugation. (Right) ReACT
strategies for methionine-based protein functional-
ization. Oxaziridine (Ox) compounds serve as oxidant-
mediated reagents for direct functionalization by
converting methionine to the corresponding sulfimide
conjugation product. During this redox process, the
Ox ReACT reagents are reduced to benzaldehyde.
(B) Model redox conjugation reaction with 25 mM of
N-acetyl-L-methionine methyl ester (S1) and 27.5 mM
of various oxaziridine compounds as substrates in
cosolvent (CD3OD/D2O = 1:1). The reactions were
monitored by detecting the chemical shift of the
methionine methyl group with 1H NMR (fig. S1).
The reaction time was 10 min in 100% CD3OD
solution due to slow reaction rate and 20 min in
5% CD3OD /D2O solution due to poor solubility of
substrate in aqueous solution. (C) The proposed reac-
tion mechanism between methionine and oxaziridine

Downloaded from [Link] on October 01, 2023

compound proceeds by nucleophilic attack of sul-
fide at N atom or O atom of oxaziridine ring, fol-
lowed by N–O bond cleavage to generate reaction
intermediate A or B, [Link] NTP or OTP is
generated, along with the corresponding aldehyde
or imine as side product, through an intramolecular
rearrangement. (D) Number of unique ReACT-sensitive
Met, Lys, and Cys residues detected in HeLa cell
lysates when treated with 1 mM Ox4 for 10 min.
(E) Yield of conjugation reaction was performed
with 15 mM of BSA carrying four methionines per
protein and 100 mM Ox4 at the indicated time point
as measured by in-gel fluorescence imaging. Error
bars, mean ± SD from three independent exper-
iments. Representative fluorescent gel is shown in
fig. S4.

reagent for methionine functionalization over its to whole-proteome level under mild biocompat- protein using a two-step labeling protocol, BSA at a
sulfur congener. As a further demonstration of the ible conditions. Finally, we tested the chemical concentration of 15 mM was first treated with
high selectivity of ReACT for methionine conjuga- stability of the sulfimide methionine conjugation 100 mM oxaziridine probe Ox4 bearing a bio-
tion, we next identified sites of probe labeling within product, finding that this linkage showed reason- orthogonal alkyne group and then subsequently
a whole proteome using liquid chromatography– able stability to acidic and basic conditions, as well coupled to Cy3-azide through a copper-catalyzed
tandem mass spectrometry (LC-MS/MS) analysis. as treatment with a strong protein disulfide re- azide-alkyne cycloaddition (CuAAC) reaction. The
HeLa cell lysates were treated with Ox4, trypsin ducing agent such as tris(2-carboxyethyl)phosphine resulting redox conjugation yield to BSA was an-
digested, and then analyzed by LC-MS/MS for (TCEP) (fig. S3). The product is stable to exposure alyzed by in-gel fluorescence imaging. ReACT pro-
probe modification on all nucleophilic amino acids to elevated temperatures (80°C) for short times ceeds rapidly and can be completed with a yield
using the X!Tandem program (32). We observed (1 hour), but prolonged exposure (18 hours) can >95% within 1 to 2 min, with 50% of labeling
labeling of 235 methionine residues and a single lead to substantial decomposition (fig. S3). occurring within the first 5 s after the addition of
lysine residue, with no other modifications de- Ox4 to the protein under standard reaction con-
tected on cysteine side chains or other nucleo- ReACT-based bioconjugation to ditions (Fig. 1E and fig. S4). Moreover, the mea-
philic amino acids (Fig. 1D and Data S1). These methionines in model protein substrates sured second-order rate constant for reaction of
experiments demonstrate the fast kinetics of the We then evaluated ReACT as a method for site- methionine (at 5- to 40-fold excess) with Ox2 in
ReACT strategy as well as near-perfect selectivity selective methionine conjugation of proteins. Start- phosphate-buffered saline is 18.0 ± 0.6 M−1 s−1,
for methionine residues from the single-protein ing with bovine serum albumin (BSA) as a model which is comparable to what is observed for the

Lin et al., Science 355, 597–602 (2017) 10 February 2017 2 of 6

R ES E A RC H | R E S EA R C H A R T I C LE

using the antibody fragment to green fluorescent

protein (GFP-Fab) as a starting model. We noted
that although the Fab scaffold possesses one
native methionine residue on its light chain
and two native methionine residues on its heavy
chain, none of these side chains are surface ac-
cessible and thus were not labeled by ReACT,
R even with high oxaziridine probe loadings (Fig.
O
O
NH 3A and fig. S8). As such, because these native
100 S N methionines are buried within the hydrophobic
Relative Abundance (%)

WT protein N N R S
H
MS = 16790 Da O interior core, ReACT offers a potentially valuable
80
strategy for Fab bioconjugation because there is
R=function group
60 Ox2 no background labeling of the wild-type Fab and
WT-Calmodulin 9 Mets
MS+9ΔM2 subsequent engineering of surface-accessible me-
40
thionine sites can enable precise antibody func-
20 Ox4 tionalization at directed locations. Using the
MS+9ΔM4
0 THIOMab platform (34), we demonstrated this
MS+8ΔM2+O possibility by replacing heavy chain (HC)–A114
Ox5
Ox2 MS+9ΔM5 or light chain (LC)–V205 residues with methi-
O
Ox2 onine and showed efficient labeling with ReACT
MS+8ΔM3+O N N ΔM3=86
Ox6
O H (fig. S8). In addition, we established rapid, near-
Ox4 WT+9ΔM6 O
quantitative, and site-specific C-terminal labeling
Ox4 with ReACT on a GFP-Fab bearing a C-terminal
MS+8ΔM5+O

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N N
O H ΔM4=96 methionine (GFP-Fab-CM) (Fig. 3, A and B). The
Ox5 resulting azide-carrying GFP-Fab (GFP-Fab-N3)
O
Ox5
MS+8ΔM6+O N N
O retained similar binding affinity to the GFP lig-
O H ΔM5=140
and compared to the wild-type Fab (fig. S9). Click
Ox6 O reactions enabled further functionalization of
Ox6
16500 17000 17500 18000 18500 N N
H
N3
ΔM6=141
GFP-Fab-N3 with biotin, fluorophore, and drug
O
Molecular mass (Da) payloads (Fig. 3B). Moreover, the resulting con-
jugates were compatible with biological environ-
Fig. 2. The ReACT strategy for protein functionalization. (A) General two-step procedure for ments. For example, we used a human embryonic
methionine-specific protein functionalization is a combination of ReACT and click reactions. Various pay- kidney 293T (HEK-293T) cell line with a doxycy-
loads (red sphere) can be installed through methionine conjugation at a directed position on a given protein. cline (Dox)–inducible cell surface GFP expression
(B) Redox conjugation of a CaM model protein (100 mM) with various Ox compounds (1 mM). The chemical system, where Dox treatment results in expres-
structures of oxaziridine probes are shown with molecular weight changes (DM) listed for the corresponding sion of GFP localized to the cell surface. Upon
[Link] deconvoluted MS data of full protein peaks are plotted in the same [Link] major peaks pre-addition of Dox followed by incubation with
correspond to CaM protein carrying nine sulfimide modifications (DM). For Ox2-labeled protein: expected Cy3-labeled GFP-Fab made by ReACT, we ob-
mass 17,564 Da, found 17,565 Da; Ox4-labeled protein: expected mass 17,654 Da, found 17,654 Da; Ox5- served excellent colocalization of Cy3 and GFP
labeled protein: expected mass 18,050 Da, found 18,051 Da; Ox6-labeled protein: expected mass 18,059 Da, signals in live HEK-293T cells. In contrast, no
found 18,060 Da. The minor peaks correspond to CaM protein bearing eight sulfimide modifications (DM) Cy3 signal was observed in control cells without
and one sulfoxide modification (O). Dox addition (Fig. 3C). Furthermore, the intensity
of the Cy3 signal was stable for at least 14 days in
CuAAC reaction (fig. S5). We then moved on to ap- alkyne- and azide-containing oxaziridine probes the presence of 100% fetal bovine serum (fig. S10).
ply ReACT to modification of calmodulin (CaM) as (1 mM) with a 100-mM CaM model protein (Fig. 2B). Taken together, these data demonstrate that
a model protein with redox-sensitive methionines LC-MS/MS results showed that ReACT enabled ReACT can enable antibody functionalization
(33). CaM carries nine redox-active methionine near quantitative installation of these bioorthog- at directed positions with a wide variety of pay-
residues, and upon pretreatment with increasing onal handles on all nine native methionine resi- loads and simultaneously retain their function
concentrations of hydrogen peroxide, we observed dues within 10 min of labeling time at room for ligand binding.
the expected dose-dependent decrease in Ox4 la- temperature, with a 25:1 selectivity over the We next moved on to apply ReACT to a thera-
beling as these residues were oxidized from me- only other observed minor CaM product bearing peutic conjugate, Herceptin-Fab (Her-Fab). As
thionine to methionine sulfoxide, the latter of eight sulfimide modifications (NTP) and one expected, ReACT did not label wild-type Her-Fab,
which is insensitive to ReACT (fig. S6). Only me- sulfoxide modification (OTP) (Fig. 2B). Further owing to its lack of surface-accessible methio-
thionine residues were identified carrying the functionalizations with biotin, fluorophore, and nines (fig. S11). By engineering Her-Fab platforms
desired modification by LC-MS/MS analysis of Ox4- polyethylene glycol (PEG) payloads proceeded carrying one or two methionine residues at the
labeled calmodulin, with no probe-generated con- smoothly (fig. S7). The high methionine reac- C terminus of the light chain, ReACT afforded
jugation modification observed on any other ami- tivity and specificity of ReACT coupled with the near quantitative conjugation with one or two
no acids (fig. S6). The results presage the potential ready availability of click reaction partners redox modifications, respectively (Fig. 3D). The
application of ReACT to probe redox-sensitive provides a straightforward method for precise data establish that ReACT can enable synthesis
methionines by distinguishing them from their protein functionalization based on this naturally of ADCs with a defined drug-to-antibody ratio
oxidized forms. occurring amino acid (Fig. 2A). (DAR) in excellent purity, which remains a major
With these data in hand, we envisioned that challenge for bioconjugation methods employ-
ReACT could enable installation of various payloads Application of ReACT to ing cysteine or lysine ligation. Moreover, the bio-
onto a protein of interest at defined methionine antibody bioconjugation orthogonal azide or alkyne handle introduced by
sites, serving as a method for functionalization To showcase the utility of ReACT in a pilot bio- methionine conjugation can be readily function-
using naturally occurring amino acids (Fig. 2A). logical application, we turned our attention to alized with additional payloads. The ADC synthe-
To this end, we evaluated the reactivity of various the synthesis of antibody-drug conjugates (ADCs), sized by linking monomethyl auristatin E (MMAE)

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R ES E A RC H | R E S EA R C H A R T I C LE

to Her-Fab exhibited a fivefold increase in tox- of cells with low, medium, and high levels of Of particular interest are the hyperreactive me-
icity to Her2-positive BT474 breast cancer cells ReACT probe Ox4 (Fig. 4A and fig. S12), we thionine targets, because they can predict sites of
compared with either wild-type Her-Fab or a mix- sought to identify hyperreactive methionines that methionine-regulated protein function. This un-
ture of wild-type Her-Fab and free MMAE (Fig. 3E), should be enriched with low-dose labeling along biased ReACT approach not only enables char-
demonstrating its utility in a biological context. with less-reactive methionine sites. By perform- acterization of previously studied redox-sensitive
ing parallel TOP-ABPP (n = 2 for all three groups) methionines in whole-proteome settings but more
Application of ReACT to in HeLa cell lysates, we were able to identify 116 importantly identifies new functional methionine
chemoproteomic identification (low dose), 458 (medium dose), and 1111 (high sites. As a positive control, we identified three
of functional methionines in cells dose) peptides that carry the desired ReACT me- hyperreactive methionines within actin, includ-
Finally, we turned our attention to the use of thionine modification (Fig. 4, A and B, and Data ing Met44 and Met47, whose redox activities
ReACT as a methionine-targeted warhead for S2). Compilation of the hyperreactive methionine- have been previously shown to play a central role
chemoproteomics applications, owing to its high containing target proteins identified in the low- in controlling actin polymerization in living cells
specificity and reactivity, as well as the small dose ReACT-treated group spanned many protein (Fig. 4C) (24, 25). With these data validating the
warhead size of the oxaziridine group (e.g., the classes, including enzymes, chaperones, and nu- ReACT method in hand, we moved on to identify
molecular weight of Ox4 is 202 Da) that allows cleoproteins, as well as many structural proteins and characterize new targets with methionine-
access to a broad range of proteins. To this end, (Data S2). In general, only surface-accessible me- dependent function. As one representative exam-
we applied ReACT to probe reactive methionines thionine residues were identified even with the ple, we found three hyperreactive methionine
in the proteome through tandem orthogonal high-dose probe, indicating that ReACT does not residues on enolase, a central enzyme in the an-
proteolysis–activity–based protein profiling (TOP- disrupt or denature proteins under these label- cient and conserved metabolic pathway of gly-
ABPP) (35). Through dose-dependent treatment ing conditions (Fig. 4C and fig. S13). colysis (36), which is important in regulating

Downloaded from [Link] on October 01, 2023

Fig. 3. ReACT for synthesis of methionine-targeted
antibody conjugates. (A) Crystal structure of 100
LC-4 GFP-Fab-CM
Her-Fab [Protein Data Bank (PDB) 1n8z] with three HC-100c

Relative Abundance (%)

GFP-Fab-N3
HC-82 80
native methionine residues shown as green sticks GFP-Fab-Biotin
and sulfur atoms shown as yellow [Link] sulfur GFP-Fab-Cy3
atoms appear to be buried in the pocket of Fab on the 60 GFP-Fab-MMAE
crystal structure. (B) The ratio of probe/methionine
40
for Fab labeling is 10, with 10-min labeling time at
room temperature. The deconvoluted MS data of
20
GFP-Fab constructs with or without ReACT label-
ing. GFP-Fab-CM: expected mass 47158 Da, found
0
47158 Da; GFP-Fab-N3 (labeled by Ox6): expected 46500 47500 48500 49500
mass 47299 Da, found 47299 Da; biotin-functionalized C-terminus Molecular mass (Da)
GFP-Fab-N3 (GFP-Fab-Biotin): expected mass 48019 Da,
found 48019 Da; Cy3-functionalized GFP-Fab-N3
(GFP-Fab-Cy3): expected mass 48282 Da, found
48282 Da. MMAE-functionalized GFP-Fab-N3 (GFP-Fab-
MMAE): expected mass 48949 Da, found 48949 Da.
+ Dox
(C) Fluorescence colocalization imaging of GFP-Fab-
Cy3 with cell surface–targeted GFP in HEK-293T
cells. The GFP was inducibly expressed on the cell GFP Cy3 Merge BF
surface with addition of 1 mM/mL of Dox before add-
ing GFP-Fab-Cy3. Cells without addition of Dox were
used as a control and show no antibody staining. All
images use the same scale bar: 20 mm. (D) The de-
convoluted MS data of Her-Fab constructs with or - Dox
without ReACT labeling. Her-Fab carries one C-
terminal methionine (Her-Fab1): expected mass
47544 Da, found 47544 Da; Ox6-labeled Her-Fab1 GFP Cy3 Merge BF
(Her-Fab1-N3): expected mass 47685 Da, found
47686 Da; Her-Fab carries two C-terminal methi-
onine (Her-Fab2): expected mass 47676 Da, found 100 100 Her-Fab2
Her-Fab1
47677 Da; Ox6-labeled Her-Fab2 (Her-Fab2-N3): ex-
Relative Abundance (%)

Her-Fab1-N3 Her-Fab2 + Drug

pected mass 48958 Da, found 47958 Da. (E) In vitro 80 80 ADC
Her-Fab2
Cell Survival (%)

cytotoxicity of Her-Fab2 [median effective concen- Her-Fab2-N3

tration (EC50) = 0.086 ± 0.02 mg/mL], noncovalent 60 60
mixture of Her-Fab2 and free MMAE (EC50 = 0.096 ±
0.04 mg/mL), and the ReACT-derived ADC from 40 40
Her-Fab2 and MMAE (EC50 = 0.015 ± 0.007 mg/mL).
Error bars, mean ± SD from three independent 20 20
experiments. EC50 values and EC50 ± SDs were de-
termined using four-parameter logistic fitting. 0 0
47000 47500 48000 48500 -3 -2 -1 0 1 2
Molecular mass (Da) Log (Concentration), µg/mL

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R ES E A RC H | R E S EA R C H A R T I C LE

diseases such as cancer via the Warburg effect more detail, we cloned and purified a yeast homo- revealed that both the turnover number (Kcat)
(37). In this scenario, cancer cells predominantly log for in vitro biochemistry studies. Treatment and the Michaelis constant (Km) are affected
produce more energy compared with nontumor- of the wild-type yeast enolase 1 with hypochlorite in the wild type upon oxidation but that these
igenic cells by manipulating glycolysis enzymes decreased enzymatic activity with concomitant values for the M171L mutant remain the same
(e.g., enolases). Among these three methionine oxidation of methionine residues, including Met171, (fig. S15). To show the physiological consequen-
residues in enolase, Met169 residue is highly on the protein (Fig. 4E). A similar decrease in ces of this methionine-based redox regulation at
conserved from yeast to mammals (correspond- protein activity was observed upon oxidation the cellular level, we generated yeast strains with
ing to Met171 on yeast enolase 1) (Fig. 4D and fig. of the M371L mutant. In contrast, the activity a double enolase 1 and enolase 2 knockout back-
S14). Moreover, this residue is close to the en- of the M171L mutant was unaffected by oxidant ground (38) and reintroduced either wild-type
zyme active site and can be oxidized along with treatment under the same conditions (Fig. 4E), enolase 1 or the M171L mutant. As depicted in
other methionine residues in the mammalian pro- suggesting that this highly conserved residue is Fig. 4F, we observed that the strain carrying
tein upon oxidant treatment (Fig. 4D and fig. S15). critical for redox regulation of enolase function. the M171L mutation is more resistant to oxida-
To characterize the functional importance of Kinetics measurements of the wild type and M171L tive stress–induced cell death compared to the
this oxidation-sensitive methionine in enolase in mutants with and without oxidant treatment strain with wild-type enolase 1, establishing that

Fig. 4. Chemoproteomic methionine profiling

with ReACT. (A) Reactive methionine profiling
with ReACT involves treatment of proteomes with
low, medium, and high doses of Ox4 probe, fol-
lowed by CuAAC-based installation of acid cleav-
able biotin-azide tag, enrichment with streptavidin
magnetic beads, and sequential on-bead trypsin
digestions to afford probe-labeled peptides for

Downloaded from [Link] on October 01, 2023

LC-MS/MS analysis. (B) The number of peptides
carrying the desired ReACTmodification on methi-
onine and appearing in two independent runs from
the low-, medium-, and high-dose groups is shown.
(C) Reactive methionine map on actin (PDB 3byh),
with hyperreactive methionines colored in red, in-
cluding Met44 and Met47, medium-reactive methi-
onines colored in purple; and less-reactive methionines
colored in blue. The methionines colored in yellow
represent residues identified by LC-MS/MS that do
not carry redox [Link] domains carrying
these yellow-colored methionines are involved in
actin polymerization. Because of this activity, no
desired modification is detected on these residues
even when these are surface accessible on the pro-
tein x-ray crystal structure. (D) Protein structure align-
ment of human alpha enolase (yellow; PDB 2psn)
and yeast enolase 1 (red; PDB 2AL1), with con-
served methionine residues shown in stick repre-
sentation. (E) The relative activity of yeast enolase 1
variants with or without treatment of NaClO (100 mM).
Error bars, mean ± SD from four independent ex-
periments. P values indicated in the figure repre-
sent results of an unpaired t test. (F) Growth curve
of wild-type (WT) (ENO2 null) and ENO1-M171L
(ENO2 null) strains, with or without treatment of
NaClO (100 mM). Knockout and mutation strains
were generated by clustered regularly interspaced
short palindromic repeats (CRISPR)–Cas9–mediated
genome editing. Error bars, mean ± SD from three
independent experiments.

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this methionine residue serves a functional redox- 6. S. Bondalapati, M. Jbara, A. Brik, Nat. Chem. 8, 407–418 (2016). 33. J. R. Erickson et al., Cell 133, 462–474 (2008).
active role in vivo. 7. E. Weerapana et al., Nature 468, 790–795 (2010). 34. J. R. Junutula et al., Nat. Biotechnol. 26, 925–932
8. S. Mizukami, Y. Hori, K. Kikuchi, Acc. Chem. Res. 47, 247–256 (2008).
Conclusion (2014). 35. A. E. Speers, B. F. Cravatt, J. Am. Chem. Soc. 127,
9. T. F. Brewer, F. J. Garcia, C. S. Onak, K. S. Carroll, C. J. Chang, 10018–10019 (2005).
To close, ReACT provides a unique and general Annu. Rev. Biochem. 84, 765–790 (2015). 36. L. A. Fothergill-Gilmore, P. A. M. Michels, Prog. Biophys. Mol.
redox-based approach to chemoselective methi- 10. I. M. Serafimova et al., Nat. Chem. Biol. 8, 471–476 (2012). Biol. 59, 105–235 (1993).
11. J. M. Ostrem, U. Peters, M. L. Sos, J. A. Wells, K. M. Shokat, 37. M. G. Vander Heiden, L. C. Cantley, C. B. Thompson, Science
onine conjugation that complements the wealth Nature 503, 548–551 (2013). 324, 1029–1033 (2009).
of acid-base conjugation methods for modifica- 12. P. Agarwal, C. R. Bertozzi, Bioconjug. Chem. 26, 176–192 (2015). 38. P. G. Leonard et al., Nat. Chem. Biol. 12, 1053–1058
tion of more nucleophilic amino acids such as 13. D. K. Nomura, M. M. Dix, B. F. Cravatt, Nat. Rev. Cancer 10, (2016).
cysteine, lysine, and serine. The relative rarity of 630–638 (2010). 39. E. A. Kabat, T. T. Wu, H. Bilofsky, J. Biol. Chem. 252,
14. L. E. Sanman, M. Bogyo, Annu. Rev. Biochem. 83, 249–273 6609–6616 (1977).
methionine in surface-accessible forms, such as (2014).
in the complementarity-determining region of 15. D. Leung, C. Hardouin, D. L. Boger, B. F. Cravatt, AC KNOWLED GME NTS
antibodies (39) that show highly similar sequence Nat. Biotechnol. 21, 687–691 (2003). We thank the U.S. Department of Energy/Lawrence Berkeley
homology, presages the potential utility of this 16. O. Boutureira, G. J. L. Bernardes, Chem. Rev. 115, 2174–2195 National Laboratory (101528-002 to C.J.C. and DE-AC02-05CH11231
(2015). to F.D.T.) for financial support, as well as NIH (GM79465 to
method for functionalization of antibodies and 17. C. Zhang et al., Nat. Chem. 8, 120–128 (2016). C.J.C.) for pilot protein labeling studies. C.J.C. is an Investigator of
their fragments as well as other proteins using 18. E. V. Vinogradova, C. Zhang, A. M. Spokoyny, B. L. Pentelute, the Howard Hughes Medical Institute and a Canadian Institute for
naturally occurring amino acids that are native S. L. Buchwald, Nature 526, 687–691 (2015). Advanced Research Senior Fellow. We also thank NIH for grant
or readily introduced by standard site-directed 19. T. H. Wright et al., Science 354, aag1465 (2016). 1S10OD020062-01 in financial support of University of California
20. A. Yang et al., Science 354, 623–626 (2016). (UC) Berkeley QB3 mass spectrometry facilities. We thank A. Killilea
mutagenesis. More broadly, the ReACT method 21. H. G. Gundlach, S. Moore, W. H. Stein, J. Biol. Chem. 234, (UC Berkeley Tissue Culture Facility) and M. Salemi and B. Phinney
enables installation of various payloads onto ther- 1761–1764 (1959). (UC Davis Proteomics Core) for expert technical assistance, as well as
apeutic and imaging proteins at well-defined 22. J. R. Kramer, T. J. Deming, Chem. Commun. (Camb.) 49, S. Pollock for the HEK-293T-GFP cell line, V. Yu for yeast genome
positions and with excellent DAR. Finally, the 5144–5146 (2013). editing, and D. Nomura for helpful discussions. A.M.W. is a Merck
23. A. Kaya, B. C. Lee, V. N. Gladyshev, Antioxid. Redox Signal. 23, Fellow of the Helen Hay Whitney Foundation. P.S.L is supported
selectivity of the oxaziridine warhead offers a 814–822 (2015).

Downloaded from [Link] on October 01, 2023

by a postdoctoral fellowship from NIH (F32CA203152). The
chemical platform for identifying and studying 24. R.-J. Hung, C. W. Pak, J. R. Terman, Science 334, 1710–1713 experimental data sets used in this study are available as
functional methionines in whole proteomes, as (2011). supplementary materials. S.L., X.Y., F.D.T., and C.J.C. are listed
well as a starting point for therapeutic interven- 25. B. C. Lee et al., Mol. Cell 51, 397–404 (2013). as inventors on a patent application describing redox-active
26. J. Moskovitz et al., Proc. Natl. Acad. Sci. U.S.A. 98, reagents for methionine conjugation.
tions based on reactive methionine activation 12920–12925 (2001).
and/or inhibition. 27. A. K. Lewis et al., Nat. Chem. Biol. 12, 860–866
(2016). SUPPLEMENTARY MATERIALS
RE FE RENCES AND N OT ES 28. D. A. Shannon, E. Weerapana, Curr. Opin. Chem. Biol. 24, [Link]/content/355/6325/597/suppl/DC1
1. M. H. Stipanuk, Annu. Rev. Nutr. 24, 539–577 (2004). 18–26 (2015). Materials and Methods
2. R. L. Levine, L. Mosoni, B. S. Berlett, E. R. Stadtman, Proc. Natl. 29. V. Bizet, C. M. M. Hendriks, C. Bolm, Chem. Soc. Rev. 44, Figs. S1 to S26
Acad. Sci. U.S.A. 93, 15036–15040 (1996). 3378–3390 (2015). Data S1 and S2
3. N. M. Giles et al., Chem. Biol. 10, 677–693 (2003). 30. J. Vidal et al., Chem. Eur. J. 3, 1691–1709 (1997). References (40–68)
4. C. E. Paulsen, K. S. Carroll, Chem. Rev. 113, 4633–4679 31. A. Armstrong, I. D. Edmonds, M. E. Swarbrick, Tetrahedron
(2013). Lett. 44, 5335–5338 (2003). 2 November 2016; accepted 11 January 2017
5. C. D. Spicer, B. G. Davis, Nat. Commun. 5, 4740 (2014). 32. R. Craig, R. C. Beavis, Bioinformatics 20, 1466–1467 (2004). 10.1126/science.aal3316

Lin et al., Science 355, 597–602 (2017) 10 February 2017 6 of 6

 Redox-based reagents for chemoselective methionine bioconjugation
Shixian Lin, Xiaoyu Yang, Shang Jia, Amy M. Weeks, Michael Hornsby, Peter S. Lee, Rita V. Nichiporuk, Anthony T.
Iavarone, James A. Wells, F. Dean Toste, and Christopher J. Chang

Science 355 (6325), . DOI: 10.1126/science.aal3316

Targeting proteins at the other sulfur

As the only amino acid with a thiol (SH) group, cysteine is easily targeted for site-selective protein modifications.
Hydrophobic methionine also has sulfur in its side chain, but its capping methyl group has hindered analogous
targeting efforts. Lin et al. introduce a complementary protocol to tether new substituents exclusively to methionine,
even in the presence of cysteine. They used an oxaziridine group as an oxidant to form sulfimide (S=N) linkages. The
approach allowed antibody-drug conjugation and chemoproteomic screening for reactive methionine surface residues.

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