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Journal of the American Chemical Society
Finally, in the course of our investigations we noticed
ACKNOWLEDGMENT
1
that the pan antiꢀKcr antibody crossꢀreacted with acetyꢀ
lated nucleosomes (Figure 3c). In principle, this crossꢀ
reactivity could complicate biochemical or proteomics
studies relying on this reagent. We reasoned that treatꢀ
ment of crotonylated histones, but not their acetylated
counterparts, with TCEP should selectively mask the
‘mark’ from antibody detection, due to the drastic
changes in steric bulk and charge that accompanies the
reaction. Indeed, treatment of crotonylated nucleosomes
with TCEP blocked the recognition of the crotonyl mark
by the antiꢀKcr antibody, whereas the ability of the pan
antiꢀKac antibody to recognize acetylated nucleosomes
was unaffected by TCEP treatment (Figure 3c, Figure
S13). Thus, TCEP can be used in simple control reacꢀ
tions to verify that signals obtained from immunoblotꢀ
ting originate from the crotonylation mark. Based on this
idea, it should be possible to selectively elute crotonylꢀ
ated proteins following immunoꢀprecipitation of cellular
extracts using antiꢀKcr antibodies (or indeed other Kcr
binding factors). Thus, we imagine that the TCEPꢀ
crotonyl reaction will prove useful in the context of exꢀ
isting antibodyꢀbased proteomics workflows. 25
The authors thank Zachary Brown, Robert Thompson and
Felix Wojcik for valuable discussions. The work was supꢀ
ported by the U.S. National Institutes of Health (NIH grants
GM107047 and CA196539).
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REFERENCES
(1) Chen, T.; Dent, S. Y. R. Nat. Rev. Genet. 2014, 15, 93ꢀ106.
(2) Jenuwein, T.; Allis, C. D. Science 2001, 293, 1074ꢀ1080.
(3) Verdin, E.; Ott, M. Nat. Rev. Mol. Cell Biol. 2015, 16, 258–
264.
(4) Shahbazian, M. D.; Grunstein, M. Annu. Rev. Biochem. 2007,
76, 75ꢀ100.
(5) Tan, M.; Luo, H.; Lee, S.; Jin, F.; Yang, J. S.; Montellier, E.;
Buchou, T.; Cheng, Z.; Rousseaux, S.; Rajagopal, N.; et al.; Cell
2011, 146, 1016–28.
(6) Chen, Y.; Sprung, R.; Tang, Y.; Ball, H.; Sangras, B.; Kim, S.
C.; Falck, J. R.; Peng, J.; Gu, W.; Zhao, Y. Mol. Cell. Proteomics
2007, 6, 812ꢀ819
(7) Dai, L.; Peng, C.; Montellier, E.; Lu, Z.; Chen, Y.; Ishii, H.;
Debernardi, A.; Buchou, T.; Rousseaux, S.; Jin, F.; et al.; Nat. Chem.
Biol. 2014, 10, 365ꢀ370
(8) Xie, Z.; Dai, J.; Dai, L.; Tan, M.; Cheng, Z.; Wu, Y.; Boeke, J.
D.; Zhao, Y. Mol. Cell. Proteomics 2012, 11, 100ꢀ107.
(9) Tan, M.; Peng, C.; Anderson, K. A.; Chhoy, P.; Xie, Z.; Dai,
L.; Park, J.; Chen, Y.; Huang, H.; Zhang, Y.; et al.; Cell Metab. 2014,
19, 605–617.
(10) Xie, Z.; Zhang, D.; Chung, D.; Tang, Z.; Huang, H.; Dai, L.;
Qi, S.; Li, J.; Colak, G.; Chen, Y.; et al.; Mol. Cell 2016, 62, 194ꢀ206.
(11) Sabari, B. R.; Tang, Z.; Huang, H.; YongꢀGonzalez, V.;
Molina, H.; Kong, H. E.; Dai, L.; Shimada, M.; Cross, J. R.; Zhao, Y.;
et al. Mol. Cell 2015, 58, 203ꢀ215.
(12) Sabari, B. R.; Zhang, D.; Allis, C. D.; Zhao, Y. Nat. Rev. Mol.
Cell Biol. 2016, 18, 90ꢀ101.
(13) Wei, W.; Mao, A.; Tang, B.; Zeng, Q.; Gao, S.; Liu, X.; Lu,
L.; Li, W.; Du, J. X.; Li, J.; et al.;. J. Proteome Res. 2017, 16, 1743ꢀ
1752.
(14) Wu, Q.; Li, W.; Wang, C.; Fan, P.; Cao, L.; Wu, Z.; Wang, F.
J. Proteome Res, 2017. 16, 3664ꢀ3671.
(15) Li, Y; Sabari, B. R.; Panchenko, T.; Wen, H.; Zhao, D.; Guan,
H.; Wan, L.; Huang, H.; Tang, Z.; Zhao, Y.; et al.; Mol. Cell 2016, 62,
181ꢀ193.
(16) Xiong, X.; Panchenko, T.; Yang, S.; Zhao, S.; Yan, P.; Zhang,
W.; Xie, W.; Li, Y.; Zhao, Y.; Allis, C. D.; Li, H. Nat. Chem. Biol.
2016, 12, 1111ꢀ1118.
(17) Andrews, F. H.; Shinsky, S. A.; Shanle, E. K.; Bridgers, J. B.;
Gest, A.; Tsun, I. K.; Krajewski, K.; Shi, X.; Strahl, B. D.;
Kutateladze, T. G. Nat. Chem. Biol. 2016, 12, 396–398.
(18) Qi, Y. K.; Chang, H. N.; Pan, K. M.; Tian, C. L.; Zheng, J. S.
Chem. Commun. 2015, 51, 14632–14635.
(19) Fan, Y. C.; Kwon, O. Chem. Commun. 2013, 49, 11588ꢀ
11619.
(20) Stewart, I. C.; Bergman, R. G.; Toste, F. D. J. Am. Chem.
Soc., 2003, 125, 8696–8697.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The crotonyl group bears a unique structure among
known protein PTMs in that it contains an α,β unsatuꢀ
rated functionality in conjugation with a carbonyl group.
Here we exploit the serendipitous discovery that this
PTM undergoes a conjugate addition reaction with the
reducing agent TCEP to give a stable adduct. This unexꢀ
pected reactivity of TCEP was traced to the presence of
its pendent carboxylate groups, which we speculate staꢀ
bilize the phosphonium species in the product. Based on
this insight, we developed a sensitive and selective
chemical probe of protein crotonylation and demonstrate
its utility in the context of proteomics efforts and mechꢀ
anistic biochemical studies. The new probe compleꢀ
ments existing antibodyꢀbased tools for studying croꢀ
tonylation.5 For example, the ability to covalently attach
a biotin moiety to the modified protein would allow for
the adoption of rigorous enrichment protocols within
proteomics workflows employing readily available
streptavidin reagents. Moreover, the chemical probe is
unlikely to be sensitive to the local sequence or PTM
environment around the crotonyl mark, and is thus exꢀ
pected to be less susceptible to epitope occlusion effects
compared to antibodies.26 Thus, we imagine that our
chemical probe will find utility in the study of this imꢀ
portant PTM.
(21) Krezel, A.; Latajka, R.; Bujacz, G. D.; Bal, W. Inorg. Chem.
2003, 42, 1994–2003.
(22) Cline, D. J., Redding, S. E., Brohawn, S. G., Psathas, J. N.,
Schneider, J. P., Thorpe, C. Biochem. 2004, 43, 15195ꢀ15203.
(23) Enders, D.; Saint‐Dizier, A.; Lannou, M. I.; Lenzen, A. Eur.
J. Org. Chem. 2006, 29ꢀ49.
(24) Nguyen, U. T. T.; Bittova, L.; Müller, M. M.; Fierz, B.; Daꢀ
vid, Y.; HouckꢀLoomis, B.; Feng, V.; Dann, G. P.; Muir, T. W. Nat.
Methods 2014, 11, 834–40.
(25) Wingren, C. Adv. Exp. Med. Biol. 2016, 926, 163–179.
(26) Clayton, A. L; Hazzalin, C. A.; Mahadevan, L. C., Mol. Cell.
2006, 23, 289ꢀ296.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on
the ACS Publications website. Full methods and experiꢀ
mental data, including Figures S1ꢀS18 (PDF).
AUTHOR INFORMATION
*muir@princeton.edu
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