S. Niwayama et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5698–5702
5701
12. The procedure for synthesis of 13C7-iodoacetanilide is as follows: 13C-labeled
iodoacetic acid (475 mg, 2.5 mmol, purchased from Cambridge Isotope
Laboratories, Inc.) was dissolved in ethyl acetate (5 mL), and the mixture was
cooled to 0 °C in an ice bath. To this solution, 13C6-labeled aniline (250 mg,
2.5 mmol, purchased from Cambridge Isotope Laboratories, Inc.) was added,
and dicyclohexylcarbodiimide (520 mg, 2.5 mmol) in ethyl acetate (5 mL) was
relatively higher molecular weight, which typically show a greater
number of isotopic peaks.21
Acknowledgements
added slowly by stirring.
A white precipitate formed immediately after
addition of the dicyclohexylcarbodiimide. The mixture was stirred at 0 °C for
30 min, and then at room temperature for an hour. The dicyclohexylurea was
removed by celite filtration, and the filtrate was evaporated to dryness and
purified by silica gel column chromatography (CH2Cl2–CHCl3 = 1/1 then
CH2Cl2) to afford the product (645 mg, 96% yield). The product was
recrystallized from CHCl3 (a yellowish solid, mp 143–144 °C). 1H NMR
(300 MHz, acetone-d6) d 9.54 (1H, br s), 6.82–7.97 (5H, m), 3.93 (2H, d,
1J*CH = 151.8); 13C NMR (75 MHz, acetone-d6) 167.0, 140.1, 129.9, 124.9, 120.2,
0.9, HRMS m/z calcd for 12C13C7H8INO (M+H)+: 268.9967, found: 268.9966.
13. PEP 13, 31 and 60 were purchased from Sigma–Aldrich, Japan, and Biomer
Technology, CA, USA. The sequences of amino acids for these peptides are
identical to those of peptides we reported earlier and referred to as MAT 13, 31,
and 60, respectively.
This work is supported by the Texas Tech University-Texas Tech
University Health Sciences Center Joint Initiative Grant. We thank
the Center for Biotechnology and Genomics, Texas Tech University,
for allowing us to use the research facilities. We also thank Ms.
Yuka Kaneko of Osaka University Graduate School of Medicine
and Wako Pure Chemical Industries, Ltd. for her technical
assistance.
References and notes
14. The typical procedures are as follows: The 0.6 mM stock solutions of the peptides
were prepared in Tris–HCl buffer (pH 9, concentration of Tris–HCl = 50 mM),
and the 20 mM stock solutions of IAA and 13C7-IAA were also prepared in
1. For example: (a) Pan, S.; Aebersold, R.; Chen, R.; Rush, J.; Goodlett, D. R.;
McIntosh, M. W.; Zhang, J.; Brentnall, T. A. J. Proteome Res. 2009, 8, 787; (b)
Rivera-Monroy, Z.; Bonn, G. K.; Guttman, A. Curr. Org. Chem. 2008, 12, 424; (c)
Gevaert, K.; Impens, F.; Ghesquiere, B.; Van Damme, P.; Lambrechts, A.;
Vandekerckhove, J. Proteomics 2008, 8, 4873; (d) Mayya, V.; Han, D. K. Exp. Rev.
Proteomics 2006, 3, 597; (e) Julka, S.; Regnier, F. J. Proteome Res. 2004, 3, 350; (f)
Goshe, M. B.; Smith, R. D. Curr. Opin. Biotechnol. 2003, 14, 101; (g) Aebersold, R.;
Mann, M. Nature 2003, 422, 198.
2. For example: (a) Hebeler, R.; Oeljeklaus, S.; Reidegeld, K. A.; Eisenacher, M.;
Stephan, C.; Sitek, B.; Stuehler, K.; Meyer, H. E.; Sturre, M. J. G.; Dijkwel, P. P.;
Warscheid, B. Mol. Cell. Proteomics 2008, 7, 108; (b) Palmblad, M.; Mills, D. J.;
Bindschedler, L. V. J. Proteome Res. 2008, 7, 780; (c) Gouw, J. W.; Tops, B. B. J.;
Mortensen, P.; Heck, A. J. R.; Krijgsveld, J. Anal. Chem. 2008, 80, 7796; (d)
Hathout, Y.; Flippin, J.; Fan, C.; Liu, P.; Csaky, K. J. Proteome Res. 2005, 4, 620; (e)
Wu, C. C.; MacCoss, M. J.; Howell, K. E.; Matthews, D. E.; Yates, J. R., III Anal.
Chem. 2004, 76, 4951; (f) Gu, S.; Pan, S.; Bradbury, E. M.; Chen, X. J. Am. Soc.
Mass Spectrom. 2003, 14, 1; (g) Veenstra, T. V.; Martinovic, S.; Anderson, G.;
Pasa-Tolic, L.; Smith, R. J. Am. Soc. Mass Spectrom. 2000, 11, 78; (h) Oda, Y.;
Huang, K.; Cross, F. R.; Cowburn, D.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 6591; (i) Ong, S.-E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen,
H.; Pandey, A.; Mann, M. Mol. Cell. Proteomics 2002, 1.5, 376.
DMSO. For the alkylation reaction, the peptide stock solution (2
13C7-IAA stock solution (2
L) were mixed and left for one hour at room
temperature. To this mixture, 2
lL) and IAA or
l
l
L of b-mercaptoethanol (BME, 25 mM) was
added to stop the reaction. The IAA–peptide and 13C7-IAA–peptide solutions
prepared in this way were mixed in the molar ratios of IAA/13C7-IAA = 0.3, 1, 2,
3, 4, 6, and 9. These mixtures were diluted with 50% acetonitrile, 0.1% TFA and
TM
subjected to MALDI MS analysis by MALDI TOF/TOF 4800 plus (Applied
Biosystems). The MS spectra were acquired automatically in the positive mode
and a total of 1000 shots were accumulated per spectrum. The mass range was
selected between 600 and 4000 m/z. Five data points were collected for each
ratio, and the S/N values of the monoisotopic peaks of IAA or 13C7-IAA modified
peptides were used for calculation of the relative ratios. The averages of these
data were plotted on the graph.
15. The modification of BSA was performed as reported previously.9 The stock
solution of BSA was prepared at a concentration of 0.1 mg/mL in a buffer
containing 100 mM Tris–HCl, 3% SDS and 20 mM tributylphosphine (TBP), and
the pH was adjusted to 8.5. To complete the reduction, this mixture was left for
one hour at room temperature. The BSA solution (10
lL) was incubated with
1
lL of IAA (200 mM in DMSO) or 1
l
L of 13C7-IAA (200 mM in DMSO) and was
3. For example: (a) Mirgorodskaya, O. A.; Kozmin, Y. P.; Titov, M. I.; Körner, R.;
Sönksen, C. P.; Roepstorff, P. Rapid Commun. Mass Spectrom. 2000, 14, 1226; (b)
Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C. Anal. Chem. 2001, 73,
2836; (c) Sakai, J.; Kojima, S.; Yanagi, K.; Kanaoka, M. Proteomics 2005, 5, 16; (d)
Stewart, I. I.; Thomson, T.; Figeys, D. Rapid Commun. Mass Spectrom. 2001, 15,
2456; (e) Hood, B. L.; Lucas, D. A.; Kim, G.; Chan, K. C.; Blonder, J.; Issaq, H. J.;
Veenstra, T. D.; Conrads, T. P.; Pollet, I.; Karsan, A. J. Am. Soc. Mass Spectrom.
2005, 16, 1221.
shaken and kept in a dark place for two additional hours. The IAA–BSA and
13C7-IAA–BSA solutions prepared accordingly were mixed in the molar ratios of
IAA/13C7-IAA = 0.35, 1, 2, 3, 4, 6, and 9. The solution was mixed with 2
lL of a
solution containing 20% (v/v) glycerol and 0.1% (w/v) bromophenol blue, and
was directly subjected to 1D SDS–PAGE, and the separation was performed at
120 V for 90 min. The gel was stained with Coomassie Brilliant Blue
overnight.The purified BSA spot was excised and subjected to in-gel
digestion in the following manner. The Eppendorf tubes containing the
4. For example: (a) Panchaud, A.; Hansson, J.; Affolter, M.; Rhlid, R. B.; Piu, S.;
Moreillon, P.; Kussmann, M. Mol. Cell. Proteomics 2008, 74, 800; (b) Nanavati,
D.; Gucek, M.; Milne, J. L. S.; Subramaniam, S.; Markey, S. P. Mol. Cell.
Proteomics 2008, 7, 442; (c) Cantin, G. T.; Yi, W.; Lu, B.; Park, S. K.; Xu, T.; Lee,
J.-D.; Yates, J. R. J. Proteome Res. 2008, 7, 1346; (d) Zhang, J.; Zhang, L.; Zhou,
Y.; Guo, Y.-L. J. Mass Spectrom. 2007, 42, 1514; (e) Rivers, J.; Simpson, D. M.;
Robertson, D. H. L.; Gaskell, S. J.; Beynon, R. J. Mol. Cell. Proteomics 2007, 6,
1416; (f) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold,
R. Nat. Biotechnol. 1999, 17, 994; (g) Ross, P. L.; Huang, Y. N.; Marchese, J. N.;
Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniel,
S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.;
Pappin, D. J. Mol. Cell. Proteomics 2004, 3, 1154; (h) Kuyama, H.; Watanabe, M.;
Toda, C.; Ando, E.; Tanaka, K.; Nishimura, O. Rapid Commun. Mass Spectrom.
2003, 17, 1642; (i) Fedjaev, M.; Trudel, S.; Tjon-A-Pan, N.; Parmar, A.; Posner,
B. I.; Levy, E.; Nifant’ev, I.; Pshezhetsky, A. V. Rapid Commun. Mass Spectrom.
2007, 21, 2671.
5. A few studies using electrophoresis and small organic molecule-tagging have
also been reported. For example: (a) Pascuarello, C.; Sanchez, J.-C.;
Hochstrasser, D. F.; Corthals, G. L. Rapid Commun. Mass Spectrom. 2004, 18,
117; (b) Cahill, M. A.; Wozny, W.; Schwall, G.; Schroer, K.; Hölzer, K.;
Poznanovic, S.; Hunzinger, C.; Vogt, J. A.; Stegmann, W.; Matthies, H.;
Schrattenholz, A. Rapid Commun. Mass Spectrom. 2003, 17, 1283; (c) Sechi, S.;
Chait, B. T. Anal. Chem. 1998, 70, 5150; (d) Gehanne, S.; Cecconi, D.; Carboni, L.;
Righetti, P. G.; Domenici, E.; Hamdan, M. Rapid Commun. Mass Spectrom. 2002,
16, 1692; (e) Münchbach, M.; Manfredo, Q.; Miotto, Q.; James, P. Anal. Chem.
2000, 72, 4047.
protein bands were placed in
protein bands were washed with 100
Next, they were washed with 100 L of a solution H2O/acetonitrile (50/50) for
5 min at 37 °C and at 600 rpm. The latter step was repeated until the dye was
completely removed, and then the spots were incubated for one minute in
a
Thermomixer Comfort (Eppendorf). The
lL H2O for 5 min at 37 °C and at 600 rpm.
l
100
l
L of 100% acetonitrile. After removal of the acetonitrile, the protein bands
L of an
were dried for 15 min. Digestion was performed by the addition of 10
l
aqueous ammonium hydrogen carbonate (30 mM) containing 100 ng of trypsin
(Promega Sequencing Grade Modified Trypsin, Promega Corporation, Madison,
USA) at 37 °C overnight, and peptide extraction was performed as described
previously.9 The extracted peptide solution was dried by Speed Vacuum and
kept at À20 °C for further analysis by the MALDI mass spectrometer.The dried
samples were mixed with 10
0.1% TFA. The digested sample (0.5
followed by the addition of 0.5 L of a matrix consisting of 5 mg/mL of
l
L of the solution containing 50% acetonitrile and
l
L) was deposited on MALDI-plate
a
l
a-
cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile and 0.1%
trifluoroacetic acid (TFA).
16. We discussed potential reasons for not observing isotope effects previously.9,10
17. (a) Zabet-Moghaddam, M.; Niwayama, S. Abstract of Papers, 57th American
Society for Mass Spectrometry Conference on Mass Spectrometry and Applied
Topics, Philadelphia, PA, 2009; Abstract ThP 558.; (b) Zabet-Moghaddam, M.;
Niwayama, S. Presented at the 63rd Southwest Regional Meeting of the
American Chemical Society, Lubbock, TX, November 2007; paper 301.
18. For the MS/MS analysis, the five strongest precursors with the S/N values
higher than 50 in the MS spectrum were selected for further MS/MS analysis.
The MS/MS was performed with the 1 kV positive mode under the collision-
6. Niwayama, S.; Kurono, S.; Matsumoto, H. Bioorg. Med. Chem. Lett. 2001, 11,
2257.
induced dissociation conditions.
A maximum of 2000 laser shots were
accumulated per MS/MS spectrum. A combination of MS and MS/MS data
was used for a database search utilizing MASCOT (Matrix Science, V2.1). The
following parameters were used for the MASCOT database search: enzyme,
trypsin; allowed missed cleavages, 1; variable modification, oxidation of
methionine. The mass tolerance for precursors was set to 50 ppm and for MS/
MS fragment ions to 0.25 Da. The fixed modification was introduced for IAA-
modified peptides (+133 Da) and also 13C7-IAA modified peptides (+140).
19. Similar observations have been reported with the use of isotope-unlabeled
iodoacetanilide. See Ref. 5a.
7. Niwayama, S.; Kurono, S.; Matsumoto, H. Bioorg. Med. Chem. Lett. 2003, 13,
2913.
8. Niwayama, S.; Kurono, S.; Cho, H.; Matsumoto, H. Bioorg. Med. Chem. Lett. 2006,
16, 5054.
9. Kurono, S.; Kurono, T.; Komori, N.; Niwayama, S.; Matsumoto, H. Bioorg. Med.
Chem. 2006, 14, 8197.
10. Zabet-Moghaddam, M.; Kawamura, T.; Yatagai, E.; Niwayama, S. Bioorg. Med.
Chem. Lett. 2008, 18, 4891.
11. Benesch, R.; Edalji, R.; Benesch, R. E. J. Biochem. Biophys. Methods 1979, 1, 129.