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Identifying sites of protein modification by using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and on-line membrane preconcentration-capillary electrophoresis-tandem mass spectrometry

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Journal of the American Society for Mass Spectrometry

Abstract

A strategy for rapidly identifying the number and sites of chemical or posttranslational modification of proteins is described. The use of matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry to determine the molecular weight of the adducted protein as well as map the proteolytic digest of peptides offers a rapid method to screen for the possible site of adduction. To unequivocally determine the amino acid sequence of the peptide bearing the adduct as well as structurally characteize the covalent modification, the peptide mixture is subjected to membrane preconcentration-capillary electrophoresis-mass spectrometry and tandem mass spectrometry (mPC-CE-MS/MS). The high resolving separation capability of capillary electrophoresis (CE) afford a chromatograhic step that lends itself to separation of complex mixtures of peptides with minimal sample loss. The membrane preconcentration-CE cartridge allows sample loading volumes 10,000-fold greater than conventional CE. In this work the binding site of the fluorescent label acrylodan to the intestinal fatty binding protein is characterized and shown to be covalently bound at lysine-27, by using mPC-CE-MS/MS.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA

    E. Kurian

  2. Department of Biochemistry and Molecular Biology and Department of Pharmacology, Mayo Clinic, Rochester, Minnesota, USA

    F. G. Prendergast

  3. Department of Biochemistry and Molecular Biology and Biomedical Mass Spectrometry Facility, Mayo Clinic, Rochester, Minnesota, USA

    A. J. Tomlinson & M. W. Holmes

  4. Department of Biochemistry and Molecular Biology, Department of Pharmacology, and Biomedical Mass Spectrometry Facility, Mayo Clinic, Rochester, Minnesota, USA

    S. Naylor

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  1. E. Kurian
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  2. F. G. Prendergast
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  3. A. J. Tomlinson
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  4. M. W. Holmes
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  5. S. Naylor
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Kurian, E., Prendergast, F.G., Tomlinson, A.J. et al. Identifying sites of protein modification by using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and on-line membrane preconcentration-capillary electrophoresis-tandem mass spectrometry. J Am Soc Mass Spectrom 8, 8–14 (1997). https://doi.org/10.1016/S1044-0305(97)82228-3

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  • DOI: https://doi.org/10.1016/S1044-0305(97)82228-3

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