Increased proteome coverage by combining PAGE and peptide isoelectric focusing: Comparative study of gel‐based separation approaches |
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Authors: | Ilian Atanassov Henning Urlaub |
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Institution: | 1. Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, , G?ttingen, Germany;2. Bioanalytics, Department of Clinical Chemistry, University Medical Center, , G?ttingen, Germany |
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Abstract: | The in‐depth analysis of complex proteome samples requires fractionation of the sample into subsamples prior to LC‐MS/MS in shotgun proteomics experiments. We have established a 3D workflow for shotgun proteomics that relies on protein separation by 1D PAGE, gel fractionation, trypsin digestion, and peptide separation by in‐gel IEF, prior to RP‐HPLC‐MS/MS. Our results show that applying peptide IEF can significantly increase the number of proteins identified from PAGE subfractionation. This method delivers deeper proteome coverage and provides a large degree of flexibility in experimentally approaching highly complex mixtures by still relying on protein separation according to molecular weight in the first dimension. |
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Keywords: | IPGs PAGE Peptide IEF Shotgun proteomics Technology |
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