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Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip |
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| Authors: | Ayse Nur Polat Karsten Kraiczek Albert J R Heck Reinout Raijmakers Shabaz Mohammed |
| Institution: | 1. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands 2. Netherlands Proteomics Centre, Padualaan 8, 3584 CH, Utrecht, The Netherlands 3. Agilent Technologies R&D and Marketing GmbH and Company KG, Hewlett-Packard-Strasse 8, 76337, Waldbronn, Germany |
| Abstract: | Quantitative detection of phosphorylation levels is challenging and requires an expertise in both stable isotope labeling as well as enrichment of phosphorylated peptides. Recently, a microfluidic device incorporating a nanoliter flow rate reversed phase column as well as a titania (TiO2) enrichment column was released. This HPLC phosphochip allows excellent recovery and separation of phosphorylated peptides in a robust and reproducible manner with little user intervention. In this work, we have extended the abilities of this chip by defining the conditions required for on-chip stable isotope dimethyl labeling allowing for automated quantitation. The resulting approach will make quantitative phosphoproteomics more accessible. A method was developed that allows the automated, online, dimethyl labeling and TiO2 enrichment of phosphopeptides from complex samples on a three-sectioned microfluidic HPLC phosphochip. The method is shown to allow quantification over at least one order of magnitude and provides a robust approach for fully automated online quantification of phosphopeptides. |
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