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An Open Tubular CEC Column of Excellent Separation Efficiency for Proteomic Analysis
An Open Tubular CEC Column of Excellent Separation Efficiency for Proteomic Analysis
Bulletin of the Korean Chemical Society. 2014. Oct, 35(10): 3115-3118
Copyright © 2014, Korea Chemical Society
  • Received : May 25, 2014
  • Accepted : June 18, 2014
  • Published : October 20, 2014
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About the Authors
Won Jo Cheong
Shabi Abbas Zaidi
Yune Sung Kim

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Experimental
Materials. Glacial acetic acid, trifluoroacetic acid (TFA), polyethylene glycol (PEG), sodium hydrogen phosphate, sodium dihydrogen phosphate, azobisisobutyronitrile (AIBN), 4-aminostyrene, ethyleneglycol dimetnacryalte (EDMA), γ-methacryloxypropyl trimethoxysilane (MAPS), lysozyme, ribonuclease A, α-chymotrypsinogen A, myoglobin, cytochrome C, and trypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade methanol, acetonitrile (ACN), 2-propanol, acetone, and water were obtained from Mallinckrodt Baker (Phillipsburg, NJ, USA). Silica capillaries (50 mm ID, 365 mm OD) were purchased from Grace (Deerfield, IL, USA).
Instrument. CEC experiments were performed on an Agilent (Waldbronn, Germany) HP 3D CE system with a diode array detector and the Chemstation data processing software. The stock buffer solution (50 mM phosphate) was prepared in distilled water and kept in a refrigerator. Later, the pH of phosphate buffer was controlled to the desired pH followed by acetonitrile addition to get the final mobile phase. All the samples and eluents were filtered through a 0.2 μm cellulose membrane before analysis. Samples were injected hydrodynamically for 4 s under a pressure of 5 mbar. The detection wavelength was set to 214 nm. All the separations were carried out at a constant CE voltage of −20 kV and a temperature of 25 ℃ throughout. The OT-MIP column was flushed by running the eluent for about 1 h to acquire the stable baseline.
Tryptic Digest and Protein Samples. Cytochrome C 2.5 mg was mixed with trypsin 1.0 mg, 2.0 M urea 500 uL, 0.1 M ammonium bicarbonate 500 uL, and incubated for 24 h at 37 ℃. Then it was quenched with 1 mL 0.1% TFA and stored in a freezer until analysis. The protein sample was prepared by dissolving 1.0 mg each of lysozyme, ribonuclease A, α-chymotrypsinogen A, myoglobin, cytochrome C in 1.0 mL water, and stored in a freezer. Later, it was diluted 1,000 times in the mobile phase and injected.
Preparation of OT-CEC Column. The fused silica capillary was modified according to the procedure published elsewhere. 35-38 Briefly, the silica capillary of 50 cm length was treated with 1 M NaOH, washed with water, 0.1 M HCl, water, and acetone in sequence, and dried under a flow of N 2 . A solution composed of 4 μL MAPS in 1.0 mL of 6.0 mM acetic acid was filled in the capillary for 6 h and the capillary was flushed thoroughly with methanol and dried under a nitrogen flow. After some trial-and-error based optimization, a mixture was prepared for formation of OT stationary phase for separation of proteins as follows: PEG (MW 10,000) 4.9 mg, 4-aminostyrene 8.2 μL, EDMA 59 μL, AIBN 3.5 mg dissolved in 1.0 mL 9/1 (v/v) ACN/2-propanol. The mixture was sonicated for 10 min and purged with nitrogen for 10 min. A piece of silica capillary was filled with the mixture and both capillary ends were sealed with rubber plugs. The reaction was carried out for 4 h at 50 ℃, then the capillary was thoroughly flushed with ACN, 9/1 (v/v) methanol/acetic acid, 5/5 (v/v) methanol/water in sequence. The same procedure was repeated for preparation of OT-CEC columns for the tryptic digest (separation of peptides) with somewhat different formulation of reaction mixture. The optimum formulation was found as follows: PEG (MW 10,000) 9.8 mg, 4-aminostyrene 24.6 μL, EDMA 59 μL, AIBN 3.5 mg dissolved in 1.0 mL 9/1 (v/v) ACN/2-propanol.
Optimization of Separation of Tryptic Digest . In addition to the optimization of formulation of reaction mixture, the elution conditions were also optimized by varying pH and mobile phase composition.
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2013R1A2A2A01067201).
References
Ye M. , Jiang X. , Feng S. , Tian R. , Zou H. 2007 Trends Anal. Chem. 26 80 - 84    DOI : 10.1016/j.trac.2006.10.012
Govorun V. M. , Archakov A. I. 2002 Biochemistry (Moscow) 67 1109 - 1123    DOI : 10.1023/A:1020959106412
Shil Y. , Xiang R. , Horváth C. , Wilkins J. A. 2004 J. Chromatogr. A 1053 27 - 36    DOI : 10.1016/j.chroma.2004.07.044
Kubota K. , Kosaka T. , Ichikawa K. 2005 J. Chromatogr. B 815 3 - 9    DOI : 10.1016/j.jchromb.2004.10.030
D'Ambrosio C. , Arena S. , Talamo F. , Ledda L. , Renzone G. , Ferrara L. , Scaloni A. 2005 J. Chromatogr. B 815 157 - 168    DOI : 10.1016/j.jchromb.2004.07.017
Capriotti A. L. , Cavaliere C. , Foglia P. , Samperi R. , Lagana A. 2011 J. Chromatogr. A 1218 8760 - 8776    DOI : 10.1016/j.chroma.2011.05.094
Kelleher N. L. 2004 Anal. Chem. 76 196A - 203A
Reid G. E. , McLuckey S. A. 2002 J. Mass Spectrom. 37 663 - 675    DOI : 10.1002/jms.346
McQueen P. , Krokhin O. 2012 Expert Rev. Proteomics 9 125 - 128    DOI : 10.1586/epr.12.8
Moradian A. , Kalli A. , Sweredoski M. J. , Hess S. 2014 Proteomics 14 489 - 497    DOI : 10.1002/pmic.201300256
Ahmed F. E. 2009 J. Sep. Sci. 32 771 - 798
Gedela S. , Medicherla N. R. 2007 Chromatographia 65 511 - 518    DOI : 10.1365/s10337-007-0215-9
Selvaraju S. , El Rassi Z. 2012 Electrophoresis 33 74 - 88    DOI : 10.1002/elps.201100431
TomᚠR. , Kleárník K. , Foret F. 2008 J. Sep. Sci. 31 1964 - 1979    DOI : 10.1002/jssc.200800113
Horvatovich P. , Hoekman B. , Govorukhina N. , Bischoff R. 2010 J. Sep. Sci. 33 1421 - 1437    DOI : 10.1002/jssc.201000050
Donato P. , Cacciola F. , Mondello L. , Dugo P. 2011 J. Chromatogr. A 1218 8777 - 8790    DOI : 10.1016/j.chroma.2011.05.070
Rigobello-Masini M. , Penteado J. C. P. , Masini J. C. 2013 Anal. Bioanal. Chem. 405 2107 - 2122    DOI : 10.1007/s00216-012-6574-6
Li Y. , Lee M. L. 2009 J. Sep. Sci. 32 3369 - 3378    DOI : 10.1002/jssc.200900478
Yue G. , Luo Q. , Zhang J. , Wu S. , Karger B. L. 2007 Anal. Chem. 79 938 - 946    DOI : 10.1021/ac061411m
Luo Q. , Yue G. , Valaskovic G. A. , Gu Y. , Wu S. , Karger B. L. 2007 Anal. Chem. 79 6174 - 6181    DOI : 10.1021/ac070583w
Luo Q. , Rejtar T. , Wu S. , Karger B. L. 2009 J. Chromatogr. A 1216 1223 - 1231    DOI : 10.1016/j.chroma.2008.09.105
Thakur D. , Rejtar T. , Wang D. , Bones J. , Cha S. , Clodfelder-Miller B. , Richardson E. , Binns S. , Dahiya S. , Sgroi D. , Karger B. L. 2011 J. Chromatogr. A 1218 8168 - 8174    DOI : 10.1016/j.chroma.2011.09.022
Wang D. , Hincapie M. , Rejtar T. , Karger B. L. 2011 Anal. Chem. 83 2029 - 2037    DOI : 10.1021/ac102825g
Rogeberg M. , Wilson S. R. , Greibrokk T. , Lundanes E. 2010 J. Chromatogr. A 1217 2782 - 2786    DOI : 10.1016/j.chroma.2010.02.025
Huang X. , Zhang J. , Horvath C. 1999 J. Chromatogr. A 858 91 - 101    DOI : 10.1016/S0021-9673(99)00795-5
Eeltink S. , Svec F. , Frechet J. M. 2006 J. Electrophoresis 27 4249 - 4256    DOI : 10.1002/elps.200600259
Kašička V. 2012 Electrophoresis 33 48 - 73    DOI : 10.1002/elps.201100419
Kašička V. 2010 Electrophoresis 31 122 - 146    DOI : 10.1002/elps.200900442
El Rassi Z. 2010 Electrophoresis 31 174 - 191    DOI : 10.1002/elps.200900576
Nilsson C. , Birnbaum S. , Nilsson S. 2011 Electrophoresis 32 1141 - 1147    DOI : 10.1002/elps.201000645
Sun L. , Zhu G. , Yan X. , Champion M. M. , Dovichi N. 2014 J. Proteomics 14 622 - 628    DOI : 10.1002/pmic.201300295
Karenga S. , El Rassi Z. 2008 J. Sep. Sci. 31 2677 - 2685    DOI : 10.1002/jssc.200800310
Roccol A. , Aturki Z. , D’Orazio G. , Fanali S. , Šolínová V. , Hlaváček J. , Kašička V 2007 Electrophoresis 28 1689 - 1695    DOI : 10.1002/elps.200600452
Yang Y. , Boysen R. I. , Matyska M. T. , Pesek J. J. , Hearn M. T. W. 2007 Anal. Chem. 79 4942 - 4949    DOI : 10.1021/ac0622633
Zaidi S. A. , Cheong W. J. 2009 J. Chromatogr. A 1216 2947 - 2952    DOI : 10.1016/j.chroma.2008.08.015
Zaidi S. A. , Cheong W. J. 2009 Electrophoresis 30 1603 - 1607    DOI : 10.1002/elps.200800541
Zaidi S. A. , Han K. M. , Hwang D. G. , Cheong W. J. 2010 Electrophoresis 31 1019 - 1028
Zaidi S. A. , Lee S. M. , Cheong W. J. 2011 J. Chromatogr. A 1218 1291 - 1299    DOI : 10.1016/j.chroma.2010.12.117
Cheong W. J. , Yang S. H. , Ali F. 2013 J. Sep. Sci. 36 609 - 628    DOI : 10.1002/jssc.201200784