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

0477435 - ÚIACH 2018 RIV DE eng J - Journal Article
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. E-ISSN 1522-2683
Institutional support: RVO:68081715
Keywords : iTRAQ labeling * low-molecular-weight color pI markers * peptides OFFGEL fractionation
OECD category: Analytical chemistry
Impact factor: 2.569, year: 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.
Permanent Link: http://hdl.handle.net/11104/0273785