High Select™ Phosphopeptide Enrichment Kits & Reagents

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.

• Convenient—pre-formulated buffers for ease-of-use in spin-column (Fe-NTA and TiO₂) or magnetic (Fe-NTA) formats to enable parallel processing of multiple samples
• 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.