2260
S. Niwayama et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2257–2261
is practically negligible. Therefore, the molar ratio of
the peptide can be determined at a high accuracy with
this method. In order to investigate the generality of this
method, we performed further analyses using two addi-
tional synthetic peptides, MAT13 and MAT30. The
sequences, monoisotopic masses, and estimated pIs are
SDTCSSQKTEVSTVSSTQK, 2001.92 Da and 6.2 for
MAT13, and Ac-HRSTVASMHRQEAVDCLKKF-
NARRKLKGA-NH2, 3377.82 Da and 11.6 for
MAT30, respectively.
11. Matsumoto, H.; Komori, N. Methods Enzymol. 2000, 316,
492.
12. Ash, J. D.; Wiechmann, A. F.; Kurono, S.; Matsumoto,
H. Invest. Ophthamol. Vis. Sci. 2001, 42, S506.
13. Alvarez, R.; Komori, N.; Kurono, S.; Li, F.; Matsumoto,
H.; Anderson, R. E. Invest. Ophthalmol. Vis. Sci. 2001, 42,
S628.
14. Pyriadi, T. M. J. Org. Chem. 1972, 37, 4184.
15. Kanaoka, Y.; Machida, M.; Ban, Y.; Sekine, T. Chem.
Pharm. Bull. 1967, 15, 1738.
.
16. Labeled C2D5NH2 HCl was purchased from Isotech Inc.,
.
Miamisburg, OH, USA, and CD3NH2 HCl was purchased
from Aldrich, Milwaukee, WI, USA. For the solvents for
reactions, commercially available anhydrous DMF and
reagent grade acetic anhydride were used without further pur-
ification.
The results for MAT13 and MAT30 are shown in Fig-
ure 3b and c, respectively, indicating that the theoretical
and observed relative ratios for d-labeled and unlabeled
N-ethylmaleimide-modified MAT13 and MAT30 are
also well correlated (r2=0.9998 and 1.000, inclina-
tion=0.9441 and 0.9968, respectively), and practically
the same as those of MAT31. These results suggest that
the ionization efficiencies of the d-labeled and unlabeled
N-ethylmaleimide-modified synthetic peptides are the
same within the experimental error.
17. Tsou, K.-C.; Barrnett, R. J.; Seligman, A. M. J. Am.
Chem. Soc. 1955, 77, 4613.
18. The synthetic procedure for d-labeled N-ethylmaleimide,
.
1, is as follows. Under N2 atmosphere, C2D5NH2 HCl (716
mg, 8.3 mmol) was dissolved in anhydrous DMF (4 mL) at
room temperature, and triethylamine (1.15 g, 8.3 mmol) was
added dropwise. The mixture was cooled to 0 ꢀC, maleic
anhydride (811 mg, 8.3 mmol) was added, and the mixture was
stirred at room temperature. After about an hour, approxi-
mately 40 mL of chloroform and 30 mL of water were added.
The reaction mixture was extracted with chloroform (Â3) and
washed with saturated sodium chloride (Â1), and dried over
anhydrous Na2SO4. The solvent was evaporated under
reduced pressure, and the mixture was purified by silica gel
column chromatography (ethyl acetate) to afford N-(ethyl)-
maleamic acid (1.16 g, 95%).
In summary, we developed a new method for quantita-
tive analysis of a variety of peptides having about 1.5–
3.5 kDa molecular weight and a pI range of about 4–12
as a model system to quantify a protein in a complex
mixture. The application of this method toward a
complex mixture of proteins will be reported in due
course.
The N-(ethyl)maleamic acid (432 mg, 2.9 mmol) was heated
at 110 ꢀC in acetic anhydride (3 mL) in the presence of anhy-
drous NaOAc (150 mg) for 15 min. The reaction mixture was
cooled and poured into an iced, saturated aqueous NaHCO3
solution. The mixture was extracted with ether (30 mL, Â4),
washed with saturated aqueous NaCl solution, and dried over
anhydrous Na2SO4. After evaporation, the residue was pur-
ified by silica gel column chromatography (hexane/ethyl ace-
tate=5:1) to afford pure d5-N-ethylmaleimide, 1 (188 mg,
50%) as oil, which was triturated with cold hexane to form a
white solid. N-ethylmaleimide, 2, was synthesized in the same
way. However, the yield was diminished (33%) due to
instability of 2 as well as low solubility of d3-methylamine HCl
salt in DMF even in the presence of triethylamine. Spectrum
data for 1 and 2 are as follows. 1: white solid, mp43–44 ꢀC, 1H
NMR (300 MHz, CDCl3) d 6.69 (s, 2H); 13C NMR (75 MHz,
CDCl3) d 170.3, 133.8, 31.6, 12.4, IR (neat, cmÀ1) 3099, 1641,
1504 HRMS m/e calcd for C6H6D5N2O2 (M+NH4)+:
148.1212, found: 148.1211.
2: White solid, mp94 ꢀC, 1H NMR (300 MHz, CDCl3) d
6.73 (s, 2H); 13CNMR (75 MHz, CDCl3) d170.7, 134.1, 22.9.
IR (neat, cmÀ1) 3099, 1700, 1664, HRMS m/e calcd for
C5H6D3N2O2 (M+NH4)+: 132.0849, found: 132.0838.
19. The typical procedures are as follows: the 0.6 mM aqu-
eous solution of MAT31(5 mL), 10 mM Tris–HCl buffer solu-
tion (pH 7.0, 5 mL), and 1 mL of 10 mM N-methylmaleimide in
ethanol were mixed as a sample solution and incubated at
room temperature for 10 min. Then, 1 mL of a-cyano-4-
hydroxycinnamic acid (CHCA) matrix solution prepared as
below was added to 1 mL of the sample solution, and 1 mL of
this mixture was analyzed by MALDI-TOF MS.
Acknowledgements
S.N. thanks Oklahoma State University, College of Arts
and Sciences for financial support (start-up funds).
H.M. was supported by NIH (EY06595, EY12190, and
RR15564). The synthetic peptide MAT 31 was a gift
from Dr. Naoka Komori.
References and Notes
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1993, 3, 327.
2. For example, see: Proteome Research: New Frontiers in
Functional Genomics; Wilkins, M. R., Williams, K. L., Appel,
R. D., Hochstrasser, D. F., Eds.; Springer: Berlin, 1997.
3. Matsumoto, H.; Kurien, B.; Takagi, Y.; Kahn, E. S.;
Kinumi, T.; Komori, N.; Yamada, T.; Hayashi, F.; Isono, K.;
Pak, W. L.; Jackson, K. W.; Tobin, S. L. Neuron 1994, 12,
997.
4. Komori, N.; Usukura, J.; Matsumoto, H. J. Cell Sci. 1992,
102, 191.
5. Gygi, S. P.; Rochon, Y.; Franza, B. R.; Aebersold, R. Mol.
Cell. Biol. 1999, 19, 1720.
6. Matsumoto, H.; Kahn, E. S.; Komori, N. Anal. Biochem.
1998, 260, 188.
7. Oda, Y.; Huang, K.; Cross, F. R.; Cowburn, D.; Chait,
B. T. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6591.
8. Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb,
M. H.; Aebersold, R. Nature Biotechnol. 1999, 17, 994.
9. Matsumoto, H.; Kahn, E. S.; Komori, N. Novartis Foun-
dation Symposium 1999, 224, 225.
20. The spectra were obtained using MALDI-TOF MS,
Voyager Elite BioSpectrometry Research Station (Serial No.
130), equipped with a delayed extraction option (PerSeptive
Biosystems, Framingham, MA, USA) operated at the accel-
erating voltage, 20 kV; grid voltage, 75%; guide wire voltage,
0.1%; and pulse delay time, 250 ns. A pulsed nitrogen laser
10. Nishizawa, Y.; Komori, N.; Usukura, J.; Jackson, K. W.;
Tobin, S. L.; Matsumoto, H. Exp. Eye Res. 1999, 69, 195.