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Thermo Scientific™ High Select™ Phosphopeptide Enrichment Kits & Reagents
Description
Mass spectrometry (MS) is a key tool for identifying sites of protein phosphorylation and quantifying phosphorylation changes. However, MS analysis of protein phosphorylation is challenging due to the low stoichiometry, high hydrophilicity, poor ionization, and incomplete fragmentation of phosphopeptides. Given the low relative abundance of phosphorylation modifications in complex protein samples, enrichment is essential for successful MS analysis of phosphopeptides. High-Select Fe-NTA and TiO2 phosphopeptide enrichment kits complement our lysis, reduction, alkylation, and digestion reagents, along with our C18, graphite spin, and high-pH reversed-phase peptide fractionation columns to provide a complete workflow for phosphopeptide enrichment.
• High specificity—recovers phosphopeptides with > 90% selectivity
• Superior recovery—enriches more total and unique phosphopeptides than other commercially available resins
• High coverage—recovers peptides with single and multiple phosphorylation sites
• Scalable—compatible with automation, including the use of KingFisher magnetic particle processors
High-Select Fe-NTA Phosphopeptide Enrichment Kit enriches phosphopeptides from complex samples using iron-chelate resin in spin columns. Each column can enrich phosphopeptides from 0.5 to 5 mg of total protein digest as starting sample. Together with pre-formulated buffers, the improved and simplified procedure enriches up to 150 μg of phosphopeptides with greater than 90% selectivity in less than 45 minutes.
High-Select TiO2 Phosphopeptide Enrichment Kit includes 24 titanium dioxide (TiO2) spin tips with optimized buffers to facilitate preparation of enriched phosphopeptides. Each column can enrich phosphopeptides from 0.5 to 3 mg of total protein digest as starting sample. The optimized buffers and simplified procedure eliminate the need for pyrrolidine in the elution buffer and subsequent clean-up using graphite spin columns. Use of this kit results in enrichment of up to 150 μg of phosphopeptides with greater than 90% selectivity. TiO2 enriches a unique set of phosphopeptides that complements the High-Select Fe-NTA IMAC Phosphopeptide Enrichment Kit.
High-Select Fe-NTA Magnetic Phosphopeptide Enrichment Kit enriches phosphopeptides from complex samples using iron-chelate magnetic agarose beads. Each reaction is sufficient to enrich 250–1000 μg of phosphopeptides from 0.5–5 mg of total protein digest as starting sample in less than 45 minutes. Additionally, the kit may also be used to enrich for chemically modified peptides with functional groups designed for use with immobilized metal affinity chromatography resins.
High Select Fe-NTA Magnetic Agarose has been specifically designed to enrich phosphopeptides and chemically modified peptides for MS analysis. Our non-aggregating, magnetite (Fe3O4), superparamagnetic beads have exceptional uniformity for high performance using both manual and automated purification applications. The Fe-NTA Magnetic agarose is available separately to provide flexibility in experimental design.
For high resolution analysis of the enriched phosphopeptides, the recommended LC column for Nanospray Flex source is Acclaim PepMap 100 C18 LC Column (Cat. No. 164942 or 164939). For EASY-Spray source, the recommended LC column is EASY-Spray C18 LC Column (Cat. No. ES902 or ES903). The stand-alone High Select Fe-NTA Magnetic Agarose beads provide flexibility for scale and throughput and may be used in both manual and automated platforms, including KingFisher 96 and KingFisher Flex magnetic particle processors.
Specifications
Specifications
| For Use With (Equipment) | Microcentrifuge |
| Product Type | TiO2 Phosphopeptide Enrichment Kit |
| Product Line | High Select |
| Content And Storage | • TiO2 Spin Tips (24) • Centrifuge Column Adadptors (24) • Binding/Equilibration Buffer (7 mL) • Wash Buffer (1.8 mL) • Phosphopeptide Elution Buffer (7 mL) |
| Label or Dye | Unlabeled |
| Shipping Condition | Wet Ice |
| Starting Material | Protease-digested Protein |
| Final Product Type | Peptides |
| Detection Method | Mass Spectrometry |
| Format | Kit |
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Frequently Asked Questions (FAQs)
Yes, we do recommend desalting peptide samples before enrichment when using High-Select Fe-NTA and TiO2 phosphopeptide enrichment kits. This helps ensure optimal results by removing detergents and salts that can interfere with the enrichment process.
No. It is important to enrich with the TiO2 kit (Cat. No. A32993) first. Afterwards, the flow-through and wash fractions from this enrichment can be processed with the Fe-NTA kit (Cat. No. A32992). If this order is reversed (that is, Fe-NTA before TiO2), there will be 2 consequences as follows: 1. There will not be any significant additional recovery of peptides (maybe just a few more peptides). 2. There will be no enrichment for the multiple phosphorylated peptides, so those would be lost.
The two phosphopeptides enrichment kits, Fe-NTA and TiO2, enrich a complementary set of phosphopeptides. Our R&D has developed a Sequential enrichment of Metal Oxide Affinity Chromatography (see https://assets.thermofisher.com/TFS-Assets/CMD/posters/PO-65032-SMOAC-Phosphoproteomics-Peptides-ASMS2017-PO65032-EN.pdf and https://www.thermofisher.com/blog/learning-at-the-bench/wp-content/uploads/sites/13/2024/10/High-SelectTM-SMOAC-Protocol.pdf?CID=bid_mic_r04_jp_cp0000_pjt0000_bid00000_0so_blg_protein_analysis_mass_spectrometry_bid_ts_mbr_24065_Social_LAB) where flow-through and wash fractions from TiO2 enrichment were combined and subjected to Fe-NTA enrichment. This sequential enrichment provides impressive coverage of phosphoproteomes.
There are four differences between the Fe-NTA kit (Cat. No. 88300) and the new High-Select Fe-NTA kit (Cat. No. A32992) kit as follows: 1. The selectivity - the ratio of number of phosphopeptides over total peptides - was significantly improved to 99% with Cat. No. A32992, because the reagents were extensively optimized for the phosphopeptide selection. 2. The phosphopeptide yield was also increased to 33 µg based on quantitative colorimetric peptide assay (Cat. No. 23275). 3. The reagent is a pre-formulated format, so mixing reagent to prepare the working solution from stock solutions provided in the old kit (Cat. No. 88300) is not necessary, so it is easier to handle. 4. The enrichment protocol is optimized and streamlined, which means there are many fewer steps than with Cat. No. 88300. Thus, it takes <45 min to finish the entire protocol compared to 2 hours with the old kit (Cat. No. 88300).
Here are some tips:
- Check the expiration date in order to assure that all components are still good.
- Keep TiO2 tips protected from light during storage.
- Make sure that you are using the correct buffer component for resuspension of your sample, and that the sample is very thoroughly dried and resuspended well.
- When incubating peptide samples with the Fe-NTA kit, ensure that you avoid end-over-end rotation or vortexing; instead gently tap the column to disperse the resin.
- When removing plugs from columns, be sure to avoid pushing liquid back into the column from the plug reservoir.
- After elution, do not store the phosphopeptides in the elution buffer but immediately proceed to drying the sample before storing at -80 degrees C until mass spec analysis.