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EzWay Phos-Pep Enrichment Kit
Catalog Number K07910 K07920
Size 50T 10T
Type Kit Kit
Datasheet
 
Features
  • Characterization of phospho-peptides
  • Highly selective enrichment of phospho-peptide from non-phosphorylated complex tryptic digest of proteins
  • Facilitates the identification of phospho-peptide by mass spectrometry
 
Kit Contents
  • Phos-Pep Solution A
  • Phos-Pep Solution B
  • Washing solution: ammonium acetate buffer
  • Dissolving solution:1% phosphoric acid
  • Phosphopeptide standard: beta casein tryptic digest
 
How to Use
  1. Selective binding of phosphopeptide
  2. Precipitation of phosphopeptide complex
  3. Washing and Dissolving
 
Background
Protein phosphorylation is one of the most frequently occurred posttranslational modification and plays a critical role in cellular regulatory events. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine, and tyrosine residues. Despite the importance and widespread occurrence of this modification, identification of protein phosphorylation site is still a challenge, due to the low copy of phosphorylated proteins in cell, even when performed on highly purified protein.

Mass spectrometry has been shown to be a reliable and routine tool to identify proteins in a high throughput manner. However, the identification of phosphorylation sites by mass spectrometry is not a trivial matter and to this day is not routine. The detection of phosphopeptides by mass spectrometry in a complex mixture, such as a tryptic mass fingerprint, is a rare occurrence. This is thought to be caused by suppression of the ionization of the mainly negatively charged phosphopeptide in the presence of a large excess of nonphosphorylated peptides.

EzWay™ Phos-Pep Enrichment Kit provides highly selective enrichment of phosphopeptide from non-phosphorylated complex trytic digest of proteins and facilitates the identification of phosphopeptide by mass spectrometry. Phosphopeptide identification relies on measuring the loss of mass. Phosphopeptides tend to lose their phosphate group as phosphoric (H3PO4) or phosphorous acid (HPO3) due to metastable decay in MALDITOF, ESI (PSD)2,3 ,ion trap(CID) or as phosphorous acid (HPO3) by phosphatase.

 
Phosphopeptide enrichment from trypsin digest of phosphoprotein
Trypsin digest Enriched phosphopeptide Characterization by MALDI-TOF PSD
 
Phosphopeptide enrichment from beta casein
MALDI-TOF mass spectrum of beta casein trypsin digest (left) and enriched phosphopeptide from beta casein trypsin digest using EzWay Phos-Pep Kit (right). Blue asterisk represent the enriched phosphopeptides.