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There are several strategies that can be deployed to increase target protein identifications: utilize post-translational modification (PTM) specific affinity binding, such as phosphopeptide enrichment, or through fractionation, such as high pH reversed-phase chromatography which separates peptides by hydrophobicity.
The novel Thermo Scientific™ ActivX™ probes and enrichment kits enable the specific targeting of several enzyme classes, including kinases, GTPases, and serine hydrolases, which are covalently labeled in the active site at the protein level and enriched after digestion.
Fe-NTA Phosphopeptide Enrichment Kit | TiO2 Phosphopeptide Enrichment and Clean Up Kit | Magnetic TiO2 Phosphopeptide Enrichment Kit | High pH Reversed Phase Fractionation Kit | Kinase Enrichment Kit with ATP Probe | |
Target | Phosphopeptides | Phosphopeptides | Phosphopeptides | All peptides | ATP binding sites |
Binding/labeling mechanism | Metal chelate affinity to phosphate groups | Metal oxide affinity to phosphate groups | Metal oxide affinity to phosphate groups | Hydrophobic interaction | Biotinylated ATP analog to active site lysine |
Enrichment strategy | IMAC-Agarose resin | Spherical porous TiO2 | TiO2 coated magnetic particles | Hydrophobic polymer based resin | Immobilized streptavidin agarose resin |
Loading capacity/rxn* | 0.5–5 mg | 0.5–3 mg | 100 µg | 10–100 µg | Lyophilized |
Format | Spin column | Tip | Magnetic bead | Spin column | Spin column |
# of elution fractions | 1 | 1 | 1 | 8 | 1 |
Processing time | 45–60 min | 45–60 min | 15 min | 30–60 min | 6.5 hr + digestion |
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*Loading capacity per reaction is based on a typical mammalian cultured cell protein digest sample
The Thermo Scientific™ Pierce™ Fe-NTA Phosphopeptide Enrichment Kit effectively captures phosphopeptides with multiple phosphates.
1 | 2 | 3 | 4 | 5 | 6 | Total | |
---|---|---|---|---|---|---|---|
TiO2 | 492 | 103 | 8 | 4 | 0 | 1 | 608 |
Fe-NTA IMAC | 234 | 34 | 216 | 3 | 1 | 0 | 488 |
Overlap | 155 | 0 | 1 | 0 | 0 | 0 | 156 |
Selective enrichment of singly and multiply phosphorylated phosphopeptides with TiO2 and Fe-NTA IMAC. Average phosphopeptide enrichment results from duplicate experiments showing the number of phosphopeptides containing one or more phosphate per peptide enriched using either method. Peptide spectrum summary results were exported from Proteome Software Scaffold 3.0 and analyzed with Microsoft™ Excel™ Software.
The Thermo Scientific™ Pierce™ Fe-NTA Phosphopeptide Enrichment Kit effectively captures phosphopeptides with multiple phosphates.
1 | 2 | 3 | 4 | 5 | 6 | Total | |
---|---|---|---|---|---|---|---|
TiO2 | 492 | 103 | 8 | 4 | 0 | 1 | 608 |
Fe-NTA IMAC | 234 | 34 | 216 | 3 | 1 | 0 | 488 |
Overlap | 155 | 0 | 1 | 0 | 0 | 0 | 156 |
Selective enrichment of singly and multiply phosphorylated phosphopeptides with TiO2 and Fe-NTA IMAC. Average phosphopeptide enrichment results from duplicate experiments showing the number of phosphopeptides containing one or more phosphate per peptide enriched using either method. Peptide spectrum summary results were exported from Proteome Software Scaffold 3.0 and analyzed with Microsoft™ Excel™ Software.
For Research Use Only. Not for use in diagnostic procedures.