Fluorogenic Derivatization Reagents Suitable for Isolation and Identification of Cysteine-Containing Proteins Utilizing High-Performance Liquid Chromatography - Tandem Mass Spectrometry

Article abstract of DOI:10.1021/ac034840i

The fluorogenic derivatization reagents with a positive charge, 4-(dimethylaminoethylaminosulfonyl)-7-chloro-2,1,3-benzoxadiazole (DAABD-Cl) and 7-chloro-2,1,3-benzoxadiazole-4-sulfonylaminoethyltrimethylammonium chloride (TAABD-Cl), are proposed for use in proteomics studies. Following derivatization of protein mixtures with these reagents, a series of standard processes of isolation, digestion, and identification of the proteins were performed utilizing high-performance liquid chromatography-fluorescence detection and tandem mass spectrometry with the probability-based protein identification algorithm. Both DAABD and TAABD derivatives were detected fluorometrically at the femtomole level and showed more than 100-fold improvement in sensitivity compared to the underivatized original compounds with an electrospray ionization ion trap mass spectrometer analysis. The modification of the MASCOT database search system memorized with the fragment information of a DAABD-attached Cys residue allowed the identification of the proteolytic peptide fragments of the derivatized bovine serum albumin (BSA) with an estimated 38% sequence coverage of BSA. Utilizing DAABD-Cl as a derivatization reagent, identification of several proteins was also possible in a soluble extract of Caenorhabditis elegans (10 μg of protein). Consequently, for identification of proteins in the complex matrixes of proteins, DAABD-Cl could be a more appropriate reagent than ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate as reported previously.

Full text of DOI:10.1021/ac034840i

Anal. Chem. 2004, 76, 728-735
Fluorogenic Derivatization Reagents Suitable for
Isolation and Identification of Cysteine-Containing
Proteins Utilizing High-Performance Liquid
Chromatography-Tandem Mass Spectrometry
Mayumi Masuda,† Chifuyu Toriumi,† Tomofumi Santa,† and Kazuhiro Imai*,‡
Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and Research Institute of Pharmaceutical Sciences, Musashino
University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 200-8585, Japan
In identifying proteins from protein matrixes, two-dimensional
polyacrylamide gel electrophoresis (2D-PAGE) has been used
widely. This is then followed by proteolytic digestion. The
sequences of proteins are usually determined through tandem
mass spectrometry (MS/ MS).3-6 The separation by 2D-PAGE,
however, is not only inappropriate for use in separating extremely
acidic, basic, or hydrophobic proteins,6-8 it needs a skillful
technique to obtain reproducible results.9 Therefore, for the
quantitative analysis of the expressed proteins, different fluores-
cent cyanine dyes have been developed.10,11 Because of the
chemical properties of the dyes, protein labeling ratio with cyanine
dyes was deliberately kept under 5%. Consequently, multiple
labeling products were produced for each protein and afforded
several spots on the gel.9
The fluorogenic derivatization reagents with a positive
charge, 4 -(dimethylaminoethylaminosulfonyl)-7 -chloro-
2,1,3-benzoxadiazole (DAABD-Cl) and 7-chloro-2,1,3-benz-
oxadiazole-4-sulfonylaminoethyltrimethylammonium chlo-
ride (TAABD-Cl), are proposed for use in proteomics
studies. Following derivatization of protein mixtures with
these reagents, a series of standard processes of isolation,
digestion, and identification of the proteins were per-
formed utilizing high-performance liquid chromatography-
fluorescence detection and tandem mass spectrometry
with the probability-based protein identification algorithm.
Both DAABD and TAABD derivatives were detected
fluorometrically at the femtomole level and showed more
than 1 0 0 -fold improvement in sensitivity compared to the
underivatized original compounds with an electrospray
ionization ion trap mass spectrometer analysis. The
modification of the MASCOT database search system
memorized with the fragment information of a DAABD-
attached Cys residue allowed the identification of the
proteolytic peptide fragments of the derivatized bovine
serum albumin (BSA) with an estimated 3 8 % sequence
coverage of BSA. Utilizing DAABD-Cl as a derivatization
reagent, identification of several proteins was also possible
in a soluble extract of Ca en or h a bd itis elega n s (1 0 µg
of protein). Consequently, for identification of proteins in
the complex matrixes of proteins, DAABD-Cl could be a
more appropriate reagent than ammonium 7-fluoro-2,1,3-
benzoxadiazole-4 -sulfonate as reported previously.
Multidimensional liquid chromatography (LC) has been used
to separate the enzymatically digested peptides followed by
identification by MS/ MS.12-15 Gygi et al. proposed a method using
(1) Chen, G.; Gharib, T. G.; Huang, C. C.; Taylor, J. M.; Misek, D. E.; Kardia,
S. L.; Giordano, T. J.; Iannettoni, M. D.; Orringer, M. B.; Hanash, S. M.;
Beer, D. G. Mol. Cell. Proteomics 2 0 0 2 , 1, 304-313.
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R. Mol. Cell. Proteomics 2 0 0 2 , 1, 323-333.
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L.; Zvelebil, M. J.; Stein, R. C.; Waterfield, M. D.; Davies, S. C.; O’Hare, M.
J. Proc. Natl. Acad. Sci. U.S.A. 1 9 9 9 , 96, 12589-12594.
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Wittmann-Liebold, B.; Svasti, J. Proteomics 2 0 0 2 , 2, 706-712.
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K.; Pleissner, K. P.; Otto, A. Muller, E. C.; Sokolowska-Kohler, W.; Grabher,
G.; Stoffler, G. Electrophoresis 1 9 9 9 , 20, 2100-2110.
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Electrophoresis 2 0 0 0 , 21, 1104-1115.
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2077.
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Pognan, F.; Hawkins, E.; Currie, I.; Davison, M. Proteomics 2 0 0 1 , 1, 377-
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Biomed. Sci. Appl. 2 0 0 1 , 752, 281-291.
Although the conduct of a quantitative biological analysis is
accepted as a routine in gathering genomic information, the
approaches used in the analysis of DNA or mRNA do not provide
exact information on the expressed proteins in real samples.1,2
The establishment of a method for the identification of the
expressed proteins is therefore essential.
* To whom correspondence should be addressed. Tel: +81-3-5400-2640. Fax:
+81-3-5400-2640. E-mail: imai-kz@kyoritsu-ph.ac.jp.
† The University of Tokyo.
(14) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.;
‡ Musashino University.
Garvik, B. M.; Yates, J. R. Nat. Biotechnol. 1 9 9 9 , 17, 676-682.
728 Analytical Chemistry, Vol. 76, No. 3, February 1, 2004
10.1021/ac034840i CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/07/2004

isotope-coded affinity tags (ICAT) reagents, biotin and a mass-
differentiated group to quantify the proteins from the protein
mixtures. The process consists of labeling the proteins with the
reagents, proteolytic digestion, isolation of the tagged peptides
(by use of an avidin column), liquid chromatogreaphic separation,
and finally identification and quantification by MS/ MS.16-19 As an
alternative approach, tandem mass tags have been developed.20
The tandem mass tags are composed of a sensitization group of
guanidino functionality and a mass normalization group to de-
rivatize proteins. The relative abundance of proteins is measured
using MS/ MS-based detection. However, the molecular masses
of ICAT reagents and tandem mass tags are relatively high, which
might cause the less soluble derivatized proteins to be absorbed
onto the tubes and columns. It was also true for the cleavable
reagents now in use, (S)-pentafluorophenyl[tris(2,4,6-trimethox-
yphenyl)phosphonium] acetate bromide, bearing high molecular
mass.21-23 Therefore, we developed water-soluble, small-mass
derivatization reagents for proteomics studies.
In a previous paper,24 we reported a method for proteomics
studies. The protein mixtures were first derivatized with a water-
soluble fluorogenic reagent, ammonium 7-fluoro-2,1,3-benzoxa-
diazole-4-sulfonate (SBD-F),25 followed by isolation, digestion of
the modified proteins, and identification of the proteins. The
identification of these proteins was performed by utilizing high-
performance liquid chromatography (HPLC)-fluorescence detec-
tion and electrospray ionization (ESI)-MS/ MS with the probability-
based protein identification algorithm. The derivatized proteins
were highly fluorescent, with long excitation (380 nm) and
emission wavelengths (520 nm), and suitable for femtomole
detection owing to the 2,1,3-benzoxadiazole (benzofurazan) skel-
eton.26,27 In addition, the derivatives with SBD-F were hydrophilic
and suitable for their isolation by HPLC. Using the proposed
method, 11 altered proteins were identified in the islets of
Langerhans in rats pretreated with dexamethazone. By using this
method, however, the SBD-peptides after digestion with enzymes
could not be detected by MS to our satisfaction, due probably to
the negatively charged sulfonyl group in the SBD moiety.
For matrix-assisted laser desorption/ ionization (MALDI)-
MS21-23,28-32 or ESI-MS,32-37 the proteins with positive charges
were proved to be highly detectable because of their easy
Figure 1. Chemical structures of DAABD-Cl and TAABD-Cl and
the derivatization reaction for thiols.
ionization and simple fragmentation.38 Thus, the peptides were
modified to have a positively charged group attached to the
N-terminus to simplify MS/ MS spectra.29-31
In this paper, to improve the detectability of the modified
proteins in MS, we develop the two water-soluble and positively
charged fluorogenic derivatization reagents for thiols instead of
SBD-F, i.e., 4-(dimethylaminoethylaminosulfonyl)-7-chloro-2,1,3-
benzoxadiazole (DAABD-Cl) and 7-chloro-2,1,3-benzoxadiazole-4-
sulfonylaminoethyltrimethylammonium chloride (TAABD-Cl). The
chemical structures of DAABD-Cl and TAABD-Cl and their
derivatives are shown in Figure 1. The DAABD and TAABD
derivatives are positively charged in solution and proved to be
sensitively detected fluorometrically and mass spectrometrically.
The applicability of DAABD-Cl is also described for identification
of an authentic bovine serum albumin (BSA) and proteins in a
soluble extract of Caenorhabditis elegans.
EXPERIMENTAL SECTION
Material and Reagents. 4-Chlorosulfonyl-7-chloro-2,1,3-ben-
zoxadiazole and N,N-dimethylethylenediamine were obtained from
Tokyo Kasei Kogyo Co. (Tokyo, Japan). Aminoethyltrimethylam-
(15) Wolters, D. A.; Washburn, M. P.; Yates, J. R., 3rd. Anal. Chem. 2 0 0 1 , 73,
5683-5690.
(16) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R.
Nat. Biotechnol. 1 9 9 9 , 17, 994-999.
(17) Han, D. K.; Eng, J.; Zhou, H.; Aebersold, R. Nat. Biotechnol. 2 0 0 1 , 19, 946-
951.
(28) Broberg, S.; Broberg, A.; Duus, J. O. Rapid Commun. Mass Spectrom. 2 0 0 0 ,
14, 1801-1805.
(29) Peters, E. C.; Horn, D. M.; Tully, D. C.; Brock, A. Rapid Commun. Mass
(18) Zhang, R.; Sioma, C. S.; Wang, S.; Regnier, F. E. Anal. Chem. 2 0 0 1 , 73,
Spectrom. 2 0 0 1 , 15, 2387-2392.
5142-5149.
(30) Spengler, B.; Luetzenkirchen, F.; Metzger, S.; Chaurand, P.; Kaufmann, R.;
Jeffery, W.; Bartlet-Jones, M.; Pappin, D. J. C. Int. J. Mass Spectrom. 1 9 9 7 ,
169, 127-140.
(19) Griffin, T. J.; Han, D. K.; Gygi, S. P.; Rist, B.; Lee, H.; Aebersold, R.; Parker,
K. C. J. Am. Soc. Mass Spectrom. 2 0 0 1 , 12, 1238-1246.
(20) Schlosser, A.; Lehmann, W. D. J. Mass Spectrom. 2 0 0 0 , 35, 1382-1390.
(21) Shen, T. L.; Huang, Z. H.; Laivenieks, M.; Zeikus, J. G.; Gage, D. A.; Allison,
J. J. Mass Spectrom. 1 9 9 9 , 34, 1154-1165.
(31) Huang, Z. H.; Shen, T. L.; Wu, J. A.; Gage, D. A.; Watson, J. T. Anal. Biochem.
1 9 9 9 , 268, 305-317.
(32) Stults, J. T.; Lai, J.; McCune, S.; Wetzel, R. Anal. Chem. 1 9 9 3 , 65, 1703-
1708.
(22) Huang, Z. H.; Wu, J.; Roth, K. D. W.; Yang, Y.; Gage, D. A.; Watson, J. T.
Anal. Chem. 1 9 9 7 , 69, 137-144.
(23) Adamczyk, M.; Gebler, J.; Wu, J. Rapid Commun. Mass Spectrom. 1 9 9 9 ,
13, 1413-1422.
(33) Cech, N. B.; Enke, C. Mass Spectrom. Rev. 2 0 0 1 , 20, 362-387.
(34) Yoshino, K.; Takao, T.; Murata, H.; Shimonishi, Y. Anal. Chem. 1 9 9 5 , 67,
4028-4031.
(24) Toriumi, C.; Imai, K. Anal. Chem. 2 0 0 3 , 75, 3725-3730.
(25) Imai, K.; Toyo’oka, T.; Watanabe, Y. Anal. Biochem. 1 9 8 3 , 128, 471-473.
(26) Imai, K.; Uzu, S.; Toyo’oka, T. J. Pharm. Biomed. Anal. 1 9 8 9 , 7, 1395-
1403.
(27) Uchiyama, S.; Santa, T.; Okiyama, N.; Toriba, A.; Imai, K. Biomed.
Chromatogr. 2 0 0 1 , 15, 295-318.
(35) Okamoto, M.; Takahashi, K. I.; Doi, T. Rapid Commun. Mass Spectrom.
1 9 9 5 , 9, 641-643.
(36) Suzuki, S.; Kakehi, K.; Honda, S. Anal. Chem. 1 9 9 6 , 68, 2073-2083.
(37) Quirke, J. M.; Van Berkel, G. J. J. Mass Spectrom. 2 0 0 1 , 36, 1294-1300.
(38) Roth, K. D.; Huang, Z. H.; Sadagopan, N.; Watson, J. T. Mass Spectrom.
Rev. 1 9 9 8 , 17, 255-274.
Analytical Chemistry, Vol. 76, No. 3, February 1, 2004 729

monium chloride was from Aldrich Co. (Milwaukee, WI). Aceto-
nitrile and methanol, both of HPLC grade, dichloromethane, and
cysteine were purchased from Kanto Chemical Co. (Tokyo, Japan).
Homocysteine, tris(2-carboxethyl)phosphine hydrochloride (TCEP),
and BSA were obtained from Sigma Chemical Co. (St. Louis, MO).
Formic acid, trifluoroacetic acid, ammonium bicarbonate, and
glutathione (reduced form) was from Wako Pure Chemicals
(Osaka, Japan). Silica gel was purchased from Merck (Darmstadt,
Germany), and guanidine hydrochloride was from Nacalai Tasque
(Kyoto, Japan). Trypsin was obtained from Promega (Madison,
WI). 3-[(3-Cholamidopropyl)dimethylammonio] propanesulfonic
acid (CHAPS) and ethylenediaminetetraacetic acid disodium salt
(Na2EDTA) were from Dojindo Laboratories (Kumamoto, Japan).
Vasopressin, oxytocin, calcitonin, and amylin were purchased from
the Peptide Institute (Osaka, Japan). All other reagents used were
of guaranteed reagent grade and used without further purification.
Apparatus. Proton nuclear magnetic resonance (1H NMR)
spectra were acquired using a JEOL LA-500 spectrometer (Tokyo,
Japan) with tetramethylsilane as an internal standard in CD3OD.
Mass spctra were obtained using an ESI ion trap mass spectrom-
eter (Esquire 3000+, Bruker Daltonics, Billerica, MA). Melting
points were measured on a Yanagimoto Micro Point Apparatus
(Tokyo, Japan). Fluorescence spectra were measured using a
Hitachi-4500 fluorescent spectrometer (Tokyo, Japan).
Synthesis of 4 -(Dim ethylam inoethylam inosulfonyl)-7 -
chloro-2 ,1 ,3 -benzoxadiazole. 4-Chlorosulfonyl-7-chloro-2,1,3-
benzoxadiazole (126.53 mg) was dissolved in CH3CN (6 mL). After
the addition of N,N-dimethylethylenediamine (88.8 mg) and
triethylamine (36 µL), the mixture was stirred at room temperature
for 10 min. The reaction mixture was evaporated to dryness under
reduced pressure, and the residue was chromatographed on silica
gel using CH2Cl2-CH3OH (9:1) to afford DAABD-Cl (120.2 mg,
87.4%) as a yellow powder: mp, 107 °C; 1H NMR δH 7.94 (1H, d,
J ) 7.5 Hz), 7.65 (1H, d, J ) 7.5 Hz), 3.06 (2H, t, J ) 6.7 Hz), 2.30
(2H, t, J ) 6.7 Hz), 2.02 (6H, s); ESI-MS m/ z 305 (M + H)+. Anal.
Calcd for C10H13O3N4SCl: C, 39.41; H, 4.30; N, 18.38. Found: C,
39.24; H, 4.40; N, 18.14.
Synthesis of 7 -Chloro-2 ,1 ,3 -benzoxadiazole-4 -sulfony-
laminoethyl Trimethylammonium Chloride. 4-Chlorosulfonyl-
7-chloro-2,1,3-benzoxadiazole (126.53 mg) was dissolved in CH3CN
(6 mL). After the addition of aminoethyltrimethylammonium
chloride (87.55 mg) dissolved in H2O (2 mL) and triethylamine
(73 µL), the mixture was stirred at room temperature for 20 min.
The reaction mixture was evaporated to dryness under reduced
pressure, and the residue was chromatographed on a column of
TSKgel ODS-80 TMG (20 × 75 mm i.d.) using the HPLC system.
The HPLC system consisted of a pump (L-7100, Hitachi, Tokyo,
Japan), a column, and an ultraviolet detector (UV-970, Jasco,
Tokyo, Japan). The UV detection was carried out at a wavelength
of 254 nm. Mobile phases: the eluent (A) 0.1% trifluoroacetic acid
and the eluent (B) water/ CH3CN/ trifluoroacetic acid (30/ 70/ 0.1).
The gradient elution was carried out from 10 to 100% (B) over 25
min with a flow rate of 3.0 mL/ min. The fraction of TAABD-Cl
including 7-chloro-2,1,3-benzoxadiazole-4-sulfonate (SBD-Cl)39 was
eluted at 19.8 min, which was collected, concentrated, and purified
to remove SBD-Cl on a column of TSKgel DEAE-5PW (75 × 2.0
mm i.d.) using the HPLC system. Mobile phases: the eluent (A)
0.1% trifluoroacetic acid and the eluent (B) water/ CH3CN/
trifluoroacetic acid (30/ 70/ 0.1). The gradient elution was per-
formed from 40 to 60% B over 10 min at a flow rate of 0.5 mL/
min. The fraction containing TAABD-Cl was eluted at 4.5 min and
evaporated to afford the trifluoroacetic acid adduct of TAABD-Cl
(127.2 mg, 58.8%) as a white powder: mp, 108-109 °C; 1H NMR
δH 8.01 (1H, d, J ) 7.3 Hz), 7.69 (1H, d, J ) 7.3 Hz), 3.46-3.48
(4H, m), 3.12 (9H, s); ESI-MS m/ z 319 (M)+. Anal. Calcd for
C13H16O5N4SClF3: C, 36.08; H, 3.72; N, 12.94. Found: C, 35.40;
H, 3.52; N, 12.52.
Time Course for the Derivatization Reaction of Thiols with
DAABD-Cl and TAABD-Cl. A 50-µL aliquot each of the fluoro-
genic reagent solution (1 mg/ mL), DAABD-Cl or TAABD-Cl in
100 mM borate buffer containing 5 mM Na2EDTA (pH 9) was
mixed with the same volume of a 10 µM mixture of cysteine,
homocysteine, and glutathione. The reaction mixture was incu-
bated at 40 °C for 10-120 min, and the reaction was stopped with
100 µL of 0.1% formic acid. After the reaction, a 5-µL aliquot of
the reaction mixture was injected into the HPLC system. The
HPLC system consisted of a pump (L-7100, Hitachi), a separation
column TSKgel 120-T QA (250 × 4.6 mm i.d.) (Tosoh, Tokyo,
Japan), and a fluorescence detector (FL-2025, Jasco). The fluo-
rescence detection was carried out at 387 and 508 nm for the
excitation and emission wavelengths, respectively. The mobile
phase was 150 mM phosphate buffer/ CH3CN (94/ 6), and the flow
rate was 1.0 mL/ min.
Measurement of Fluorescence Spectra. The solutions of
the fluorescent derivatives of cysteine, homocysteine, and glu-
tathione (10 µM) in 150 mM phosphate buffer/ CH3CN (94/ 6)
were used to obtain the fluorescence spectra and the maximum
excitation and emission wavelengths.
Fluorescence Characteristics of DAABD Derivatives. Fluo-
rescence intensities of the derivative of glutathione (20 µM) with
DAABD-Cl in various pH solutions (Britton-Robinson buffer)
were measured at maximum excitation and emission wavelengths.
Solution A (100 mL) for the Britton-Robinson buffer consisted
of 232 µL of 85% phosphoric acid, 240 µL of acetic acid, and 247
mg of boric acid. Solution B (100 mL) was 0.8 g of sodium
hydroxide in water.
Identification and Detection of DAABD and TAABD
Derivatives of Thiols Using LC-MS. The derivatization reaction
was performed with DAABD-Cl or TAABD-Cl according to the
optimized condition (see Results and Discussion). The reaction
mixture of cysteine, homocysteine, and glutathione was directly
injected into the HPLC-MS system. Chromatography was per-
formed using a HP 1090 series II system (Hewlett-Packard GmbH)
and a separation column, Cadenza TC-18 column (12-nm pores
of silica, 100 × 2.0 mm i.d.) (Imtakt, Kyoto, Japan). Mobile
phase: the eluent (A) 0.1% formic acid and the eluent (B) water/
CH3CN/ formic acid (50/ 50/ 0.1). The gradient elution was carried
out from 0 to 100% B over 15 min with a flow rate of 0.2 mL/ min.
Derivatization Reaction of Peptides and BSA with DAABD-
Cl and TAABD-Cl. A 20-µL aliquot of 250 nM peptide solution
(vasopressin, oxytocin, calcitonin, and amylin) or BSA (final
concentration 50 nM) was mixed with the same volume of the
respective 0.5-5.0 mM TCEP, 17.5 mM derivatization reagent
(DAABD-Cl or TAABD-Cl), 10 mM Na2EDTA, and 50 mM
(39) Andrews, J. L.; Ghosh, P.; Ternai, B.; Whitehouse, M. W. Arch. Biochem.
Biophys. 1 9 8 2 , 214, 386-396.
730 Analytical Chemistry, Vol. 76, No. 3, February 1, 2004

CHAPS. Each reagent was dissolved in 100 mM borate buffer (pH
9.0) containing 6.0 M guanidine. Each reaction mixture was
incubated at 40 °C for 30-120 min, and the reaction was stopped
with 200 µL of 0.1% formic acid. After the reaction, a 30-µL aliquot
of the reaction mixture was injected into the HPLC system. The
HPLC system consisted of a pump (L-7100, Hitachi), a column of
Capcellpak C8 SG 300 (30-nm pore size, 100 × 2.0 mm i.d.,
Shiseido, Tokyo, Japan), and a fluorescence detector (FL-2025,
Jasco). Fluorescence detection was carried out at 387 and 508
nm for the excitation and emission wavelengths, respectively.
Mobile phases: the eluent (A) 0.1% trifluoroacetic acid and the
eluent (B) water/ CH3CN/ trifluoroacetic acid (30/ 70/ 0.1). The
gradient elution was carried out from 0 to 100% B over 30 min
with a flow rate of 0.5 mL/ min.
Identification of BSA Derivatized with DAABD-Cl Using
HP LC and Tandem Mass Spectrometry. The eluate of the BSA
derivative was concentrated to 60 µL under reduced pressure.
Twenty microliters of the residue (∼2.0 pmol) was diluted with
180 µL of 50 mM ammonium bicarbonate solution (pH 7.8)
containing 2 µg/ mL trypsin and 1.0 mM calcium chloride, and
the resultant mixture was incubated for 2 h at 37 °C. The
ploteolytic peptide mixture was directly subjected to LC-MS/
MS using an ESI ion trap mass spectrometer.
Chromatography was performed using a HP 1090 series II
system and a column of Cadenza TC-18 column (12-nm pores
silica, 100 × 2.0 mm i.d.). Mobile phases: eluent (A) 1.0 mM
ammonium formate and eluent (B) 1.0 mM ammonium formate/
CH3CN (50/ 50). The gradient elution was carried out from 0 to
100% B over 60 min with a flow rate of 0.2 mL/ min. The
identification of the protein was performed against the NCBInr
database with the MASCOT (Matrix Science Ltd.) database-
searching algorithm memorizing the DAABD-attached thiol resi-
due of cysteine.
Derivatization and Identification of C. elega n s P roteins
with DAABD-Cl. C. elegans (strain Bristol N2) was grown on
NGM agar, using the OP50 strain of Escherichia coli as a food
source at 20 °C and separated from bacteria by flotation on M9
buffer. After washing with M9 buffer twice, the worms were stored
at -80 °C until use. Then, they were suspended with an equal
volume of 10 mM CHAPS and lysed by sonication on ice. The
soluble fraction was collected by centrifugation at 10 000 rpm for
5 min at 4 °C. The supernatant was stocked as a soluble fraction
at -20 °C. Protein concentration of the fraction was determined
by the Bradford method40 using BSA as a standard. About 20 µL
(100 µg of protein) of the supernatant was mixed with the same
volume of the respective 2.5 mM TCEP, 17.5 mM DAABD-Cl, 10
mM Na2EDTA, and 50 mM CHAPS in 100 mM borate buffer (pH
9.0) containing 6.0 M guanidine. After the reaction mixture was
incubated at 40 °C for 30 min, the reaction was stopped with 200
µL of 0.1% formic acid, and then a 30-µL aliquot of the reaction
mixture (10 µg of protein) was injected into the HPLC system.
HPLC was performed with the same condition as described above
except using a column of RP for protein (30-nm pore size, 250 ×
4.6 mm i.d.) (Imtakt). Mobile phases: the eluent (A) 0.1%
trifluoroacetic acid and the eluent (B) water/ CH3CN/ trifluoro-
acetic acid (70/ 30/ 0.1). The gradient system was from 70 to 30%
B over 100 min with a flow rate of 0.25 mL/ min.
Figure 2. Synthetic routes for DAABD-Cl and TAABD-Cl.
For the identification, several peak fractions of the fluorescent
protein derivatives were isolated and concentrated to 10 µL under
reduced pressure. Each fraction was diluted with 90 µL of 50 mM
ammonium bicarbonate solution (pH 7.8) containing 2 µg/ mL
trypsin and 1.0 mM calcium chloride, and the resultant mixture
was incubated for 2 h at 37 °C. Each ploteolytic peptide mixture
was directly subjected to LC-MS/ MS using an ESI ion trap mass
spectrometer. The chromatographic conditions are the same as
described above in Identification of BSA Derivatized with DAABD-
Cl Using HPLC and Tandem Mass Spectrometry.
RESULTS AND DISCUSSION
Design and Synthesis of DAABD-Cl and TAABD-Cl. As
described in the introduction, the benzofurazan reagents used in
the present work have a moiety of positive charge instead of
negative charge as in SBD-F. Therefore, the sulfonic acid moiety
in SBD-F was replaced with a dimethyl- or trimethylaminoethy-
laminosulfonyl moiety. Two corresponding new reagents, DAABD-
Cl and TAABD-Cl, were synthesized. As shown in Figure 2, the
structures of these reagents are similar to 4-aminosulfonyl-7-fluoro-
2,1,3-benzoxadiazole (ABD-F)41 and 4-(N,N-dimethylaminosulfo-
nyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F).42 Since, in our pre-
liminary experiment, ABD-F and DBD-F were more reactive than
SBD-F, both ABD-F and DBD-F decomposed in the presence of
reducing agent (TCEP),43 whereas SBD-F did not. Therefore, we
planned to make the reagents with the less reactive chlorine atom
instead of fluorine atom at the 7 position of the benzofrazan
structure in DAABD and TAABD structures, and thus, DAABD-
Cl and TAABD-Cl were designed. As expected, they were
nonfluorescent in aqueous organic solvent such as water/ CH3-
CN or water/ CH3OH.
Reaction Conditions and Reactivity of the Derivatization
Reaction of DAABD-Cl and TAABD-Cl for Thiols. Time course
studies on the derivatization reaction of small molecular mass
thiols (cysteine, homocysteine, glutathione) with DAABD-Cl or
TAABD-Cl were performed at 40 °C (pH 9.0). As shown in Figure
3, the fluorescence emerged and its intensity reached the
maximum at 20 and 30 min for DAABD-Cl and TAABD-Cl,
(41) Toyo’oka, T.; Imai, K. Anal. Chem. 1 9 8 5 , 57, 1931-1937.
(42) Toyo’oka, T.; Suzuki, T.; Saito, Y.; Uzu, S.; Imai, K. Analyst 1 9 8 9 , 114, 413-
419.
(40) Bradford, M. M. Anal. Biochem. 1 9 7 6 , 72, 248-254.
(43) Lauren, S. L.; Treuheit, M. J. J. Chromatogr., A 1 9 9 8 , 798, 47-54.
Analytical Chemistry, Vol. 76, No. 3, February 1, 2004 731

Figure 4. pH profile of the fluorescence intensity of the derivative
of glutathione with DAABD-Cl.
Figure 3. Time course for the derivatization reactions of thiols with
(A) DAABD-Cl and (B) TAABD-Cl. Symbols: 9, cysteine; b, ho-
mocysteine; 2, glutathione.
respectively, indicating that the reagents rapidly reacted with thiols
under these mild conditions to afford fluorescence. Since SBD-F
requires 120 min for the derivatization reaction at 40 °C (data not
shown), it can be concluded that DAABD-Cl and TAABD-Cl are
more reactive than SBD-F and that DAABD-Cl was more reactive
than TAABD-Cl.
As mentioned above, as the structures of DAABD-Cl and
TAABD-Cl are similar to ABD-F and DBD-F, it was expected that
they would decompose with TCEP. However, there were no
decomposition of DAABD-Cl and TAABD-Cl in the presence of
TCEP (data not shown). Furthermore, the derivatives with these
reagents were also stable with TCEP, whereas ABD-cysteine
containing peptide was delabeled in the presence of reducing
reagent (dithiothreitol).43
Fluorescence Characteristics of Thiol Derivatives with
DAABD-Cl and TAABD-Cl. The fluorescence spectra of the thiol
derivatives showed that the maximum excitation and emission
wavelengths were respectively 387 and 508 nm for DAABD
derivatives but 386 and 507 nm for TAABD derivatives.
The relationship between the medium pH and the fluorescence
intensities of the DAABD derivative of glutathione is shown in
Figure 4. In the acidic and neutral conditions (at under pH 7),
the intensity was more than 14 times higher than those in the
basic conditions (at pH 11 and above). Thus, the fluorescence
intensity of the derivative decreases as the solution becomes more
basic. We therefore concluded that, for a more sensitive detection
of the derivatives, the detection should be performed at a pH under
7. It is plausible that the nitrogen atom at the dimethylamino group
of DAABD-Cl may be deprotonated in the basic condition and
quenching of the fluorescence occurred by an electron transfer
from nitrogen atom in the excited state of the fluorophore. The
observation is in agreement with the so-called photoinduced
Figure 5. Chromatograms obtained from the thiols derivatized with
(A) DAABD-Cl and (B) TAABD-Cl (5 pmol). Peaks: 1, cysteine; 2,
homocysteine; 3, glutathione. Chromatographic conditions are de-
scribed in the Experimental Section.
electron transfer.44,45 Accordingly, in acidic and neutral conditions,
we presumed the nitrogen atom was protonated.
Linearity and Detection Limits for DAABD and TAABD
Derivatives. The chromatograms (Figure 5) obtained from the
derivatives of cysteine, homocysteine, and glutathione with
DAABD-Cl or TAABD-Cl showed a single peak for each of the
derivatives, and no interfering peaks were observed. Neither
DAABD-Cl nor TAABD-Cl elution peaks appeared because the
reagents themselves do not fluoresce. The detection limits (signal-
to-noise ratio 3) for cysteine, homocysteine, and glutathione were
110, 62.8, and 81.2 fmol, respectively, for the DAABD derivatives
and 147, 70.4, and 85.2 fmol, respectively, for the TAABD
derivatives. The calibration curves for each derivative were linear
over the range from 100 fmol to 100 pmol, and the correlation
coefficients were greater than 0.999.
Identification and Detection of DAABD and TAABD
Derivatives of Thiols Using LC-MS. The derivatization reaction
mixtures of thiols with DAABD-Cl or TAABD-Cl were subjected
to HPLC with an ESI ion trap mass spectrometer. The mass
spectra for each derivative of DAABD-Cl or TAABD-Cl (Figure
6) showed that the derivatives of DAABD-Cl were detected as
the base ion peaks of m/ z ) 390 (M + H)+, 404 (M + H)+, and
576 (M + H)+ for cysteine (MW ) 121), homocysteine (MW )
(44) Uchiyama, S.; Santa, T.; Imai, K. Analyst 2 0 0 0 , 125, 1839-1845
(45) Onoda, M.; Uchiyama, S.; Santa, T.; Imai, K. Anal. Chem. 2 0 0 2 , 74, 4089-
4096
732 Analytical Chemistry, Vol. 76, No. 3, February 1, 2004

Figure 6. Positive-ion ESI mass spectra of the derivatized thiols (5 pmol). (A) cysteine with DAABD-Cl, (B) homocysteine with DAABD-Cl, (C)
glutathione with DAABD-Cl, (D) cysteine with TAABD-Cl, (E) homocysteine with TAABD-Cl, and (F) glutathione with TAABD-Cl.
135), and glutathione (MW ) 307), respectively. In contrast, the
derivatives of TAABD-Cl were detected as the base ion peaks of
m/ z ) 404 (M)+, 418 (M)+, and 590 (M)+ for cysteine, homocys-
teine, and glutathione, respectively.
peaks at all concentrations. Therefore, DAABD-Cl was selected
hereafter to derivatize peptides and proteins.
In the preliminary experiments, reaction temperatures higher
than 50 °C resulted in a decrease of peak intensity. Thus, 40 °C
was selected for the reaction. Likewise, in an examination of the
concentrations of DAABD-Cl, 3.5 mM was selected to achieve
complete derivatization. The reaction mixture was incubated at
40 °C for 10-120 min, and the fluorescence intensity reached the
maximum at 30 min (data not shown).
Considering the data obtained in preliminary experiments, the
following derivatization conditions were selected: 100 mM borate
buffer (pH 9.0) containing 6.0 M guanidine hydrochloride with
3.5 mM DAABD-Cl, 1.0 mM EDTA and 10 mM CHAPS at 40 °C
for 30 min. Under these conditions, the mixtures of peptides and
a protein, i.e., vasopressin, oxytocin, calcitonin, amylin, and BSA,
were derivatized without precipitation, and the chromatograms
showed a single fluorescent peak for each compound tested
(Figure 7). Good linearities were obtained between the peak areas
and the amounts of compounds from 10 to 1000 fmol (r > 0.999).
The detection limits for vasopressin, oxytocin, calcitonin, amylin,
and BSA were 7.0, 4.5, 5.0, 4.0, and 0.5 fmol, respectively.
Identification of BSA Derivatized with DAABD-Cl Using
HP LC and Tandem Mass Spectrometry. The derivatized BSA
with DAABD-Cl was digested with trypsin. A part of the resulting
The ratios of intensities of base peak ion of each derivative
with DAABD-Cl to the corresponding underivatized cysteine,
homocysteine, and glutathione were 3.0 × 103, 2.3 × 102, and 2.1
× 102, respectively, whereas in case of TAABD-Cl, the ratios were
2.0 × 103, 1.6 × 102, and 1.7 × 102 for cysteine, homocysteine,
and glutathione, respectively. In contrast, the respective ratios of
the derivatives with SBD-F were 23, 4.0, and 1.6 for cysteine,
homocysteine, and glutathione, suggesting that the derivatization
with DAABD-Cl or TAABD-Cl resulted in a significant enhance-
ment (up to 100-3000-fold) of detectability as compared with that
with SBD-F. Therefore, DAABD-Cl and TAABD-Cl are more
sensitive reagents in MS detection than SBD-F.
Derivatization Reaction of Peptides and BSA with DAABD-
Cl and TAABD-Cl. The derivatization conditions with the
reagents were investigated with BSA as a model protein. BSA was
dissolved in 6.0 M guanidine hydrochloride and 2.0 mM EDTA
(to avoid the oxidation of thiols). With DAABD-Cl (3.5 mM), the
addition of TCEP (0.1-1.0 mM) induced a dose-dependent
reaction yield, with the maximum obtained at 0.5 mM TCEP,
whereas, with TAABD-Cl, TCEP induced only some interfering
Analytical Chemistry, Vol. 76, No. 3, February 1, 2004 733

Figure 7. Representative chromatogram of several peptides and
BSA derivatized with DAABD-Cl. Peaks: 1, vasopressin (500 fmol);
2, oxytocin (500 fmol); 3, calcitonin (500 fmol); 4, amylin (500 fmol);
5, BSA (50 fmol). Chromatographic conditions are described in the
Experimental Section.
Figure 9. Chromatogram of the proteins (10 µg) in the soluble
fraction of C. elegans derivatized with DAABD-Cl. Peaks: 1, ribosomal
protein S3a (MW ) 28 942); 2, calreticulin precursor (MW ) 45 588);
3, ribosomal protein L1 (MW ) 38 635); 4, elongation factor 1-R (MW
) 50 636); 5, malate dehydrogenase (MW ) 35 098); 6, 40S
ribosomal protein (MW ) 22 044); 7, vitellogenin (MW ) 193 098);
8, arginine kinase (MW ) 41 969); 9, HSP-1 heat shock 70-kDa
protein A (MW ) 69 680), and 10, ribosomal protein L7Ae (MW )
13 992). Chromatographic conditions are described in the Experi-
mental Section.
Derivatization and Identification of C. elega n s P roteins
with DAABD-Cl. It is known that C. elegans is good material for
proteomics studies analysis because this is the first multicellular
model animal for which the genome sequence was determined,46
and for which the amino acid sequence database is available.47
So far, Kaji et al. have reported that 69 spots were separated by
2D-PAGE and identified by in-gel digestion followed by MALDI-
MS,48 and Schrimpf et al. identified 152 proteins through 2D-
PAGE.49
Figure 9 shows a chromatogram of proteins (∼10 µg) obtained
from a soluble fraction of C. elegans derivatized with DAABD-Cl.
After isolation, tryptic digestion, and LC-MS/ MS identification
of the arbitrary selected peak fractions, we identified 10 proteins:
(1) ribosomal protein S3a (MW ) 28 942); (2) calreticulin
precursor (MW ) 45 588); (3) ribosomal protein L1 (MW )
38 635); (4) elongation factor 1-R (MW ) 50 636); (5) malate
dehydrogenase (MW ) 35 098); (6) 40S ribosomal protein (MW
) 22 044); (7) vitellogenin (MW ) 193 098); (8) arginine kinase
(MW ) 41 969); (9) HSP-1 heat shock 70-kDa protein A (MW )
69 680), and (10) ribosomal protein L7Ae (MW ) 13 992).
Although, in the present experiment, only arbitrary selected 10
proteins were identified (Figure 9), the better separation on an
elaborate column could enable identification of more proteins in
a single run of the derivatization procedure.
Figure 8. Positive-ion ESI MS/MS spectrum of the peptide fragment
obtained from the digested BSA derivatized with DAABD-Cl (∼0.5
pmol, m/z ) 969.0).
mixture of peptides (∼0.5 pmol each) was separated by HPLC
and detected using a fluorescence detector or ESI ion trap mass
spectrometer to perform the MS/ MS analysis. The 25 fluorescent
peptides were detected on the HPLC chromatogram in agreement
with the 25 peaks of theoretical expectation, considering that the
derivatization with DAABD-Cl of the protein did not disturb the
conventional tryptic digestion. Figure 8 shows an example of ESI-
MS/ MS spectra, on which b- and y-type ions were predominant,
indicating that there were no significant unexpected MS fragmen-
tation. Utilizing the modified MASCOT database search system,
memorized with the fragment information of the cysteine residue
attached to DAABD (from 103 to 390), the sequence information
of 21 proteolytic peptides [9 cysteine-containing peptides (m/ z )
544.3, 589.2, 629.0, 634.3, 698.5, 828.4, 902.2, 969.0, 1060.5) and
12 non-cysteine-containing peptides] was obtained from the
derivatized BSA, which represents 232 amino acids residues, an
estimated 38% coverage of BSA. Therefore, we concluded DAABD
derivatives were applicable to HPLC-MS/ MS analysis of proteins.
In conclusion, compared to SBD-F, the reagents presented in
this work afforded a more sensitive mass spectrometric detection
(46) T. C.e. S. Consortium Science 1 9 9 8 , 282, 2012-2018.
(47) http:/ / www.sanger.ac.uk/ Projects/ C_elegans/ wormpep.
(48) Kaji, H.; Tsuji, T.; Mawuenyega, K. G.; Wakamiya, A.; Taoka, M.; Isobe, T,
Electrophoresis 2 0 0 0 , 21, 1755-1765.
(49) Schrimpf, S. P.; Langen, H.; Gomes, A. V.; Wahlestedt, T, Electrophoresis
2 0 0 1 , 22, 1224-1232.
(50) Toriumi, C.; Santa, T.; Imai, K. Proc. Japan Acad., Ser. B. 2 0 0 3 , 79, 137-
140.
734 Analytical Chemistry, Vol. 76, No. 3, February 1, 2004

of the derivatives of the protein and thus are more appropriate
reagents for proteomic studies. DAABD-Cl in particular would be
useful for the identification of proteins in the complex matrixes
of proteins. Furthermore, it could be ideal for quantitative
proteomics studies when two different reagents bearing similar
structures yet with different fluorescence characteristics, such as
benzoselenadiazole and benzothiadiazole reagents, are used. For
example, as the maximum emission (340 nm) and excitation
wavelengths (542 nm) of 7-halogeno-2,1,3-benzoselenadiazole-4-
sulfonate derivatives50 are different from those of SBD derivatives,
two different samples are derivatized with the respective reagents,
mixed together, and detected fluometrically with each of the
suitable wavelengths.
ACKNOWLEDGMENT
The authors thank Dr. Takeshi Iwatsubo and Mr. Tomoki
Kuwahara, The University of Tokyo, for supplying the worm with
kind technical guidance. We also thank Dr. Chang-Kee Lim, MRC
Bioanalytical Science Group and Dr. Chiho Lee, Yamato Scientific
Co. Ltd., for their kind suggestions and valuable discussion. We
thank Mr. Itaru Yazawa, Imtakt Co. Ltd., for his kind supply of
Cadenza TC-C18 and RP for protein columns.
Received for review July 23, 2003. Accepted November
20, 2003.
AC034840I
Analytical Chemistry, Vol. 76, No. 3, February 1, 2004 735