Combining Protein-Based IMAC, Peptide-Based IMAC, and MudPIT for Efficient Phosphoproteomic Analysis
- 26 January 2008
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of Proteome Research
- Vol. 7 (3) , 1346-1351
- https://doi.org/10.1021/pr0705441
Abstract
Immobilized metal affinity chromatography (IMAC) is a common strategy used for the enrichment of phosphopeptides from digested protein mixtures. However, this strategy by itself is inefficient when analyzing complex protein mixtures. Here, we assess the effectiveness of using protein-based IMAC as a pre-enrichment step prior to peptide-based IMAC. Ultimately, we couple the two IMAC-based enrichments and MudPIT in a quantitative phosphoproteomic analysis of the epidermal growth factor pathway in mammalian cells identifying 4470 unique phosphopeptides containing 4729 phosphorylation sites.Keywords
This publication has 26 references indexed in Scilit:
- Large-scale phosphorylation analysis of mouse liverProceedings of the National Academy of Sciences, 2007
- Automatic Validation of Phosphopeptide Identifications from Tandem Mass SpectraAnalytical Chemistry, 2007
- State-of-the-art in phosphoproteomicsProteomics, 2005
- Mass Spectrometric Contributions to the Practice of Phosphorylation Site Mapping through 2003Molecular & Cellular Proteomics, 2005
- Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling PathwayMolecular & Cellular Proteomics, 2005
- Immunoaffinity profiling of tyrosine phosphorylation in cancer cellsNature Biotechnology, 2005
- Automatic Quality Assessment of Peptide Tandem Mass SpectraBioinformatics, 2004
- Selective Isolation at the Femtomole Level of Phosphopeptides from Proteolytic Digests Using 2D-NanoLC-ESI-MS/MS and Titanium Oxide PrecolumnsAnalytical Chemistry, 2004
- Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiaeNature Biotechnology, 2002
- Signaling—2000 and BeyondCell, 2000