Prince:Phosphopeptide Enrichment: Difference between revisions
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=Brief Introduction= | =Brief Introduction= | ||
[[http://en.wikipedia.org/wiki/Phosphorylation Phosphorylation]] is the most common [http://en.wikipedia.org/wiki/Posttranslational_modification post-translational modification]. Phosphorylation changes a peptides molecular weight by about 80 mass units. This change is clearly discernible using mass spectrometry, but the negative charge on the phosphate group decreases the peptides charge state. Peptides with lower charge states | [[image:phosphorylation.jpeg|400px|frame]] [[http://en.wikipedia.org/wiki/Phosphorylation Phosphorylation]] is the most common [http://en.wikipedia.org/wiki/Posttranslational_modification post-translational modification]. Phosphorylation changes a peptides molecular weight by about 80 mass units. This change is clearly discernible using mass spectrometry, but the negative charge on the phosphate group decreases the peptides charge state and makes peptide ionization difficult. Peptides with lower charge states can appear to be machine noise, and when a peptide becomes neutral or negative it cannot be read by the mass spectrometer at all. Also, the percent of phosphorylated peptides in a cell is very low compared to the percent of dephosphorylated peptides, and the mass spectrometer only selects the most abundant peptides for MS2 identification. Therefore, phosphopeptides are rarely identified with high confidence in complex samples. Fortunately techniques have been developed to isolate phosphopeptides from complex samples. In our lab we use TiO<sup>2</sup> beads to isolate phosphopeptides. | ||
=Literature= | =Literature= | ||
1. Tichy, A.; Salovska, B.; Rehulka, P.; Klimentova, J.; Vavrova, J.; Stulik, J.; Hernychova, L., Phosphoproteomics: Searching for a needle in a haystack. J. Proteomics 2011, 74 (12), 2786-2797. | 1. Tichy, A.; Salovska, B.; Rehulka, P.; Klimentova, J.; Vavrova, J.; Stulik, J.; Hernychova, L., Phosphoproteomics: Searching for a needle in a haystack. J. Proteomics 2011, 74 (12), 2786-2797. | ||
2. Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P., Large-scale phosphorylation analysis of mouse liver. Proc Natl Acad Sci U S A 2007, 104 (5), 1488-93. | |||
3. Kettenbach, A. N.; Gerber, S. A., Rapid and Reproducible Single-Stage Phosphopeptide Enrichment of Complex Peptide Mixtures: Application to General and Phosphotyrosine-Specific Phosphoproteomics Experiments. Anal. Chem. 2011, 83 (20), 7635-7644. | |||
Revision as of 23:06, 6 February 2012
Brief Introduction
[Phosphorylation] is the most common post-translational modification. Phosphorylation changes a peptides molecular weight by about 80 mass units. This change is clearly discernible using mass spectrometry, but the negative charge on the phosphate group decreases the peptides charge state and makes peptide ionization difficult. Peptides with lower charge states can appear to be machine noise, and when a peptide becomes neutral or negative it cannot be read by the mass spectrometer at all. Also, the percent of phosphorylated peptides in a cell is very low compared to the percent of dephosphorylated peptides, and the mass spectrometer only selects the most abundant peptides for MS2 identification. Therefore, phosphopeptides are rarely identified with high confidence in complex samples. Fortunately techniques have been developed to isolate phosphopeptides from complex samples. In our lab we use TiO2 beads to isolate phosphopeptides.
Literature
1. Tichy, A.; Salovska, B.; Rehulka, P.; Klimentova, J.; Vavrova, J.; Stulik, J.; Hernychova, L., Phosphoproteomics: Searching for a needle in a haystack. J. Proteomics 2011, 74 (12), 2786-2797. 2. Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P., Large-scale phosphorylation analysis of mouse liver. Proc Natl Acad Sci U S A 2007, 104 (5), 1488-93. 3. Kettenbach, A. N.; Gerber, S. A., Rapid and Reproducible Single-Stage Phosphopeptide Enrichment of Complex Peptide Mixtures: Application to General and Phosphotyrosine-Specific Phosphoproteomics Experiments. Anal. Chem. 2011, 83 (20), 7635-7644.
