Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Scheme 1. Reactions of Selenium Reagents 1 and 2 with Thiols of Proteins/Peptides
addition of thiols onto unsaturated CdC bonds such as using
acrylate 23 or maleimide derivatives as common labeling agents. 24
These reagents have selectivity toward thiols and have been often
used in practical thiol analysis. However, the Michael-addition
product is irreversible, which cannot allow enriching and purifying
analyte compounds from complex matrices. 25 The third approach
involves thiol exchange reaction such as using 5,5′-dithiobis(2nitrobenzoic
acid) known as Ellman’s reagent; 26,27 a large excess
amount of the disulfide reagent is necessary to ensure complete
derivatization of all thiols in the sample. As a result, the newly
formed disulfides in the reaction can possibly further react with
residual thiols of the target molecule to form undesirable disulfides.
For example, Udgaonkar et al. used Ellman’s reagent in a
100-fold molar excess to label the thiol protein based on the
exchange reaction in the study of the cooperativity of a fast protein
folding reaction by MS. 27 Besides the problems mentioned above,
these derivatization reactions have other limitations, including a
long reaction time, low conversion yield, or more than one possible
site for tagging. Therefore, new derivatization chemistry suitable
for MS detection of thiol, particularly with high thiol selectivity,
fast reaction speed, good reaction reversibility, and high conversion
yield, is still needed for the structural analysis of peptides
and proteins in biological samples.
Because selenium is an essential element in vivo 28 and a key
component of selenoproteins known as antioxidant enzymes,
selenium chemistry has recently attracted increasing attention.
Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (reagent 1,
Scheme 1), has been considered as a mimetic of glutathione
peroxidase (GPx) 29-32 and an anti-inflammatory drug since being
found. 33,34 The mechanism for ebselen-catalyzed thiol oxidation
(23) Wang, G.; Hsieh, Y.; Wang, L.; Prelusky, D.; Korfmacher, W. A.; Morrison,
R. Anal. Chim. Acta 2003, 492, 215–221.
(24) Seiwert, B.; Hayen, H.; Karsta, U. J. Am. Soc. Mass Spectrom. 2008, 19,
1–7.
(25) Jalili, P. R.; Ball, H. L. J. Am. Soc. Mass Spectrom. 2008, 19, 741–750.
(26) Sevcikova, P.; Glatz, Z.; Tomandl, J. J. Chromatogr., A 2003, 990, 197–
204.
(27) Kumar Jha, S.; Udgaonkar, J. B. J. Biol. Chem. 2007, 282, 37479–37491.
(28) Yoneda, S. J.; Kazuo, T. S. Toxicol. Appl. Pharmacol. 1997, 143, 274–280.
(29) Forstrom, J. W.; Zakowski, J. J.; Tappel, A. L. Biochemistry 1978, 17, 2639–
2644.
(30) Birringer, M.; Pilawa, S.; Flohé, L. Nat. Prod. Rep. 2002, 19, 693–718.
(31) Jacob, C.; Giles, G. I.; Giles, N. M.; Sies, H. Angew. Chem, Int. Ed. 2003,
42, 4742–4758.
(32) Haenen, G. R.; De Rooij, B. M.; Vermeulen, N. P.; Bast, A. Am. Soc.
Pharmacol. Exp. Therapeut. 1990, 37, 412–422.
has been proposed: 35,36 the Se-N bond of ebselen is cleaved by
thiol RSH to produce the corresponding selenenyl sulfide Se-S,
which further reacts with excess thiol RSH to produce selenol
and disulfide compound RSSR. However, Mugesh et al. recently
reported that ebselen as a GPx-like redox catalyst is indeed
inactive, and the reaction of ebselen with PhSH only produces
the Se-S product, not disulfide PhSSPh, even with an excess
amount of PhSH. 36 Very recently, based on this novel selenium
chemistry, we synthesized fluorescent probes carrying Se-N
bonds and used them for detecting and imaging thiols in living
cells 6,37 by FL. It was shown that the FL probes capture cellular
thiols selectively in the presence of diverse species such as
inorganic metal ions, dopamine, histamine, L-adrenaline, etc.
Following that, Zhang et al. used piazselenole containing Se-N
bond to probe physiological thiols based on electrochemical
reactions. 38 Highly specific derivatization of thiols using compounds
containing Se-N bonds makes them promising candidates
to derivatize peptides and proteins for subsequent MS characterization.
Although the analysis of protein thiols and disulfide bonds
is important in proteomics applications, the selenium chemistry
received surprisingly limited attention. 39 Here we present a
systematic study of derivatization of amino acids, peptides, and
proteins using Se-N containing reagents and examine the utility
of this chemistry for analytical applications involving mass
spectrometry.
In this study, we carried out a series of reactions of two Se-N
containing reagents, ebselen and N-(phenylseleno)phthalimide
(reagent 2, Scheme 1), with various biological thiol compounds
such as cysteine, reduced peptide glutathione GSH, and �-lactoglobulin
A protein. As shown in Scheme 1, the Se-N bonds in
reagents 1 and 2 are cleaved by thiols to form new Se-S bonds
as shown in products 3 and 4, respectively (Scheme 1). Our
experimental results showed that this thiol derivatization reaction
is highly selective, rapid, reversible, and efficient (quantitative in
(33) Müller, A.; Cadenas, E.; Graf, P.; Sies, H. Biochem. Pharmacol. 1984, 33,
3235–3239.
(34) Sies, H.; Masumoto, H. Adv. Pharmacol. 1997, 38, 229–246.
(35) Mugesh, G.; du Mont, W.-W.; Sies, H. Chem. Rev. 2001, 101, 2125–2179.
(36) Sarma, B. K.; Mugesh, G. J. Am. Chem. Soc. 2005, 127, 11477–11485.
(37) Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G. J. Am. Chem. Soc.
2007, 129, 11666–11667.
(38) Wang, W.; Li, L.; Liu, S.; Ma, C.; Zhang, S. J. Am. Chem. Soc. 2008, 130,
10846–10847.
(39) Sakurai, T.; Kanayama, M.; Shibata, T.; Itoh, K.; Kobayashi, A.; Yamamoto,
M.; Uchida, K. Chem. Res. Toxicol. 2006, 19, 1196–1204.
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