Purification & Enrichment

• High capacity (at least 4 micromoles of phosphopeptide per ml of resin)
• Precharged with iron (III) as a stable chelate
• Provides single-step phosphocompound purification
• Supplied as an approximate 50% suspension in 50% glycerol buffered at pH 5.0
• Supplied with technical bulletin providing detailed procedure for use

View Protocol Diagram

Data

• Resin Enrichment - HPLC
• Resin Elution Selectivity - MALDI-MS
• Elution Conditions

Mass spectrometry (Matrix Assisted Laser Desorption/ Ionization Time of Flight (MALDI-TOF) or Electrospray Ionization (ESI)) of phosphopeptides from tryptic protein digests is a powerful tool for charactering and identification of phosphorylation sites. A combination of low intrinsic abundance, inefficient ionization, and/or signal suppression of most phosphopeptides may limit or even prevent detection, unless the phosphopeptide content is significantly enriched prior to analysis. This kit conveniently includes all materials needed to enrich for phosphopeptides from digested samples in a robust and unbiased manner (see binding comparison). The phosphopeptide capture matrix is a novel Ga⁺³ chelate silica based on a proprietary nitriloacetic acid (NTA) analog. The silica beads are approximately 20 microns in diameter with a pore size of 1,000 Angstroms. The matrix is packed into spin columns for easy, microscale affinity capture of phosphopeptides (see workflow).

The complete solution kit – 24 samples up to 25 nmoles phosphophopeptide each

• Mass spec compatible – save time, reduce sample handling and potential loss
• Proteomics Grade Trypsin - for clean and complete digests
• PHOS-Select™ Gallium Spin Columns – high capacity, novel Ga⁺³ silica media for fast, unbiased capture and recovery
• Controls – validate your process for confidence and reporting
• Buffers – enzyme reaction, binding, washing and elution optimized formulations for robust performance
• Consumable equipment included – matched equipment means no risk of sample loss, additional purchases or waste

Workflow highlighting the use of the spin columns for digestion and selective enrichment of phosphopeptides from sample.

Binding comparison: Selected portions of HPLC chromatograms demonstrating enrichment of phosphopeptides for IMAC technologies.
Standard phosphopeptides representing the three most common sites of phosphorylation phosphoserine, phosphothreonine, and phosphotyrosine were used. The lyophilized solids were first dissolved in water and an approximately equimolar mixture of the peptides was formulated. Each phosphopeptide was added to the BSA digest at a weight ratio of ~1.7% to produce a total phosphopeptide content of ~5% by weight. Quantitation results are given below. Note that competitor A, B, and C technologies were biased in selecting Peptide 2, while Sigma’s technology bound and eluted the peptides in approximately the same ratio as applied to the column.

Kit Recovery of phosphopeptide standards Specificity* 1 2 3 Total Sigma 59% 52% 74% 59% 50% Competitor A 6% 19% 11% 13% 28% Competitor B 39% 56% 17% 42% 28% Competitor C 37% 65% 37% 46% 25%

Product Number	P5726	P2850
	Tyrosine Protein Phosphatases and Acid and Alkaline Phosphatases	Ser/Thr Protein Phosphatases and Alkaline Phosphatase L-Isozymes
(--)-p-Bromotetramisole
Cantharidin
Imidazole
Microcystin LR
Sodium molybdate
Sodium orthovanadate
Sodium tartrate

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