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

• Published in: Electrophoresis Vol. 38; no. 16; pp. 2034 - 2041
• Main Authors: Michelland, Sylvie, Bourgoin‐Voillard, Sandrine, Cunin, Valérie, Tollance, Axel, Bertolino, Pascal, Slais, Karel, Seve, Michel
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01.08.2017; Wiley-VCH Verlag
• Subjects: Biochemistry; Biochemistry, Molecular Biology; Biological properties; Chemical Fractionation; Chromatography, High Pressure Liquid - methods; Color; Coloring Agents - chemistry; Computer Science; Fractionation; Humans; Hydrogen-Ion Concentration; Isoelectric focusing; Isoelectric Focusing - methods; iTRAQ labeling; Life Sciences; Low‐molecular‐weight color pI markers; Markers; Mass spectrometry; Molecular Weight; Peptides; Peptides - analysis; Peptides OFFGEL fractionation; Proteins; Proteome - analysis; Scientific imaging; Serum Albumin - analysis; Signal and Image Processing; Spectroscopy; Tandem Mass Spectrometry - methods; Technology assessment; Workflow; Low-molecular-weight color pI markers; Peptides OFFGEL fractionation; iTRAQ labeling
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.201700075

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.