Thermo Scientific Pierce TiO2 Phosphopeptide Enrichment and Clean-up Kit enables fast, selective enrichment of phosphorylated peptides for mass spectrometry using titanium dioxide spin tips, graphite spin columns and optimized buffers.
The complete kit includes 24 titanium dioxide (TiO2) spin tips and graphite spin columns with buffers to facilitate preparation of enriched and desalted phosphopeptides for analysis by mass spectrometry (MS).
- Convenient – spin-column format of titanium dioxide and graphite columns enable parallel processing and clean up of multiple samples in less than 2 hours
- High capacity – each column enriches up to 300µg of phosphopeptides
- Complementary – TiO2 enriches a unique set of phosphopeptides that complements the Pierce Fe-NTA IMAC Phosphopeptide Enrichment Kit
- High selectivity – recover phosphopeptides with >90% selectivity
The Pierce TiO2 Phosphopeptide Enrichment and Clean-up Kit protocol includes stringent washing conditions that increase the selectivity for phosphopeptides to >95%. Because some of the salts in these stringent washes may still be present in the eluted samples, the Pierce TiO2 Phosphopeptide Enrichment and Clean-up Kit includes graphite spin columns to desalt and concentrate enriched phosphopeptides, resulting in more successful phosphopeptide analysis results.
This kit is compatible with samples digested in solution or after in-gel digestion with trypsin or other MS grade proteases. Using the Pierce TiO2 Phosphopeptide Enrichment and Clean-up Kit and the Pierce Fe-NTA IMAC Phosphopeptide Enrichment Kits will enable complementary sets of phosphopeptides to be removed from complex samples.
|TiO2 and Fe-NTA IMAC Resins enrich a complementary set of phosphopeptides. The Venn diagram details the number of unique phosphopeptides identified from 250µg of peptides prepared from nocodazole-treated U2OS cells. Phosphopeptides were enriched with the Pierce TiO2 Phosphopeptide Enrichment and Clean-up Kit and the Pierce Fe-NTA IMAC Phosphopeptide Enrichment Kit. Eluted peptides were desalted and concentrated with Graphite Spin Columns before LC-MS/MS analysis on a Thermo Scientific Orbitrap XL Mass Spectrometer. Data was analyzed with Thermo Scientific Proteome Discoverer 2.1 and Proteome Software Scaffold 3.0.
Selective enrichment of singly and multiply phosphorylated phosphopeptides with Thermo Scientific Pierce TiO2 and FE-NTA IMAC resins. Average phosphopeptide enrichment results from duplicate experiments showing the number of phosphopeptides containing one to six (center columns) phosphates per peptide enriched using either resin. Peptide spectrum summary results were exported from Proteome Software Scaffold 3.0.
About the TiO2 Resins:
Titanium dioxide enrichment of phosphopeptide samples is an essential tool for comprehensive MS analysis of phosphopeptides. The unique phosphopeptide binding properties of TiO2 resin results in different selectivity and preferential enrichment of multiply phosphorylated peptides compared to immobilized chelated metal ions (IMAC). TiO2 resin typically enriches more phosphopeptides than IMAC resins, and TiO2 preferentially enriches monophosphorylated peptides.
TiO2 resin typically binds phosphopeptides with a higher affinity than IMAC and the preferential enrichment of singly phosphorylated peptides with TiO2 is likely due to difficulty in eluting multiply phosphorylated peptides. To enrich a more comprehensive set of phosphopeptides, the flow-through and washes from IMAC resin can be acidified and applied to TiO2 resins and the resulting eluates pooled for analysis. In addition, the number of phosphopeptides recovered can be significantly increased by pre-fractionation of samples with strong cation exchange chromatography to reduce sample complexity prior to enrichment with TiO2 or IMAC processing.
|TiO2 selectively enriches more phosphopeptides than Fe-NTA IMAC. The numbers refer to the total and unique number of phosphopeptides enriched by each method. The Y-axis is the percentage of phosphopeptides in the number of total and unique peptides, or the percentage of acidic residues in enriched unique phosphopeptides.
- Larsen M.R., et al. (2005). Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics. 4(7):873-86.
- Carrascal, M., et al. (2008). Phosphorylation Analysis of Primary Human T Lymphocytes Using Sequential IMAC and Titanium Oxide Enrichment. J. Proteome Res. 7(12):5167-5176.
- Wilson-Grady, J.T., et al. (2008). Phosphoproteome Analysis of Fission Yeast. J. Proteome Res. 7(3):1088-1097.
- Larsen, M.R., et al. (2004). Improved detection of hydrophilic phosphopeptides using graphite powder microcolumns and mass spectrometry: evidence for in vivo doubly phosphorylated dynamin I and dynamin III. Mol. Cell Proteomics. 3:456-465.
Mass Spectrometry Sample Prep Workflow
Sample preparation for mass spectrometry technical guide
Pierce Fe-NTA Phosphopeptide Enrichment Kit
Pierce Graphite Spin Columns
In-gel Tryptic Digestion Kit
Pierce Phosphoprotein Enrichment Kit
Pierce Magnetic Phosphopeptide Enrichment Kit
Halt Phosphatase Inhibitor Cocktails