The Thermo Scientific™ High-Select™ Fe-NTA Phosphopeptide Enrichment Kit enables efficient enrichment of phosphorylated peptides from complex and fractionated protein digests for analysis by mass spectrometry (MS). (This product replaces Cat. No. 88300.)
Features of the Fe-NTA Phosphopeptide Enrichment Kit:
• Convenient—pre-formulated buffers for ease-of-use and spin-column format to enable parallel processing of multiple samples
• High binding capacity—each column enriches up to 150 μg of phosphopeptides
• 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
The 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.
Mass spectrometry 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. Because of the low relative abundance of phosphorylation modifications in complex protein samples, enrichment is essential for successful MS analysis of phosphopeptides. The improved High-Select Fe-NTA Phosphopeptide Enrichment Kit complements 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.
The new procedure and optimized buffers have been shown to improve the selectivity and yield compared to the previous kit (Figure 1). The High-Select Fe-NTA Phosphopeptide Enrichment Kit is complementary to the High-Select™ TiO2 Phosphopeptide Enrichment Kit
, with both kits enriching similar numbers of phosphopeptides but different subpopulations of phosphorylated peptides (Figure 2).
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. ES802 or ES803).