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Low-molecular-weight color pI markers to monitor on-line the peptide focusing process in OFFGEL fractionation

  1. 1.
    0477435 - UIACH-O 2018 RIV DE eng J - Článek v odborném periodiku
    Michelland, S. - Bourgoin-Voillard, S. - Cunin, V. - Tollance, A. - Bertolino, P. - Šlais, Karel - Seve, M.
    Low-molecular-weight color pI markers to monitor on-line the peptide focusing process in OFFGEL fractionation.
    Electrophoresis. Roč. 38, č. 16 (2017), s. 2034-2041. ISSN 0173-0835
    Institucionální podpora: RVO:68081715
    Klíčová slova: iTRAQ labeling * low-molecular-weight color pI markers * peptides OFFGEL fractionation
    Kód oboru RIV: CB - Analytická chemie, separace
    Obor OECD: Analytical chemistry
    Impakt faktor: 2.569, rok: 2017

    High-throughput mass spectrometry-based proteomic analysis requires peptide fractionation
    to simplify complex biological samples and increase proteome coverage. OFFGEL
    fractionation technology became a common method to separate peptides or proteins using
    isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing
    process may be further optimized and controlled in terms of separation time and pI
    resolution. Here we evaluated OFFGEL technology to separate peptides from different
    samples in the presence of low-molecular-weight (LMW) color pI markers to visualize
    the focusing process. LMW color pI markers covering a large pH range were added to
    the peptide mixture before OFFGEL fractionation using a 24-wells device encompassing
    the pH range 3–10. We also explored the impact of LMW color pI markers on peptide
    fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by
    RP_HPLC prior to MS analysis using MALDI-TOF/TOF mass spectrometry in MS and
    MS/MS modes. Here we report the performance of the peptide focusing process in the
    presence of LMW color pI markers as on-line trackers during the OFFGEL process and
    the possibility to use them as pI controls for peptide focusing. This method improves the
    workflow for peptide fractionation in a bottom-up proteomic approach with or without
    iTRAQ labeling.
    Trvalý link: http://hdl.handle.net/11104/0273785
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