With the completion of the human genomic sequences, biological researchers are turning to the systemic analysis of biological systems.  A complete description of a biological system requires analysis on both the nucleic acid and protein levels.  Major efforts of current proteomic analysis technologies are directed toward the rapid and sensitive identification of the proteins that control biological systems.  However, rapid and accurate identification and characterization of large numbers of proteins remain significant challenges in proteomics research.  One of the most commonly used methods for protein analysis and characterization is 2D gel electrophoresis, followed by in-gel digestion and mass spectrometric analysis of the resulting peptides.  The ICAT™, isotope coded affintiy tag, method followed by 2-D HPLC allows quantitative characterization of cysteine containing peptides. Similar methods are also being developed for use on proteins in solution.  The mass spectrometric fingerprints are subsequently used in the search of sequence databases for the identification of proteins.  Success in the identification may vary with the completeness of the database, the sensitivity of the mass spectrometer, the presence of isobaric masses, post-translational modifications, etc.

Benefits of Pi³™ Peptide Reagents

• Specific Isolation

Isolation based on specific functionality
Covalent attachment to activated solid-support
Efficient wash removes non-specific adsorption
Releasing reagent easily cleaves covalent bond

• Improved Identification

Specific isolation adds constraint to data-base searches to improve accuracy
Complex mixtures are simplified
Low-level components are more easily identified

• Significant time and money savings

Ready to use kit
No methods development or optimization
Uses common laboratory reagents
Requires only vortex mixer and mini-centrifuge
Expanding line of reagents and chemistries *

• More information

Multi-component mixtures are more easily determined
Presence or absence of specific functionality can be ascertained
Modified form of target functionality usually does not  react

• Higher Productivity

Multiple samples may be run concurrently

Description

The Pi³ PhosphoTyrosine-QE Reagent Kit is the first in a family of phosphate specific reagent kits that provides an easy self-contained method, using a novel water-insoluble reagent that forms a covalent bond with phosphotyrosine peptides. The reagent allows for the selective isolation of PhosphoTyrosine peptides from peptide mixtures, or for the collective isolation of pSer, pThr and pTyr, down to the 2 percent occurrence level. These separated phosphate containing peptides subsequently may be regenerated and analyzed by mass spectrometry or liquid chromatography. Use of the mass spectrometric data and the method of digestion, when coupled with the added constraint that the peptides contain PhosphoTyrosine, can lead to improved accuracy in the identification of proteins. Chromatographic analysis of the PhosphoTyrosine peptides can simplify complex separations.

The Chemistry

The Pi³ PhosphoTyrosine-QE Reagent is a solid-phase reagent which performs the following chemistry on a phosphotyrosine peptide:

The Pi³ PhosphoTyrosine-QE Reagent is a solid-phase reagent which performs the following chemistry on pSer, pThr and pTyr peptides:

The Method

The method employed for the use of the Pi³ PhosphoTyrosine-QE Reagent consists of 5 basic steps using standard laboratory reagents:

1. Capture the phosphotyrosine peptides (or pSer, pThr and pTyr as a group) on the solid-support.
2. Wash the support to remove any non-bound peptides.
3. Release the bound phosphotyrosine peptides.
4. Collect the released peptides and wash the solid-support.
5. Desalt and concentrate the phosphotyrosine peptide solution prior to analysis.

In addition to pipettors, balances, etc., the following equipment is needed for the use with the Pi³ PhosphoTyrosine-QE Reagent Kit:

 1. Vortex mixer with micro tube insert:
     a.  The holes in the insert must be, or must be modified    to be ~.375" (10mm) in diameter
     b. If the holes in the insert are not through holes, the    depth of the holes must be ~1.75" (45mm) deep.
     c.  The Vortex Genie II, fitted with the 6" platform head and the 60 micro tube insert is recommended.

 2. Bench top centrifuge:
     a.  Capable of 10,000 RPM
     b. Capable of holding 1.5-2 ml microcentrifuge tubes

Ordering Information

Pi³™ PhosphoTyrosine-QE Reagent Kit  Part No. X-7204-4 Price: $200.00

Pi³™ PhosphoTyrosine-QE Reagent Kit  Part No. X-7204-8 Price: $350.00

Included are:
  1. Four or Eight isolation packs, respectively.
  2. One accessory pack

Capacity of each isolation pack is for a digestion of up to 5 nmol of protein.
Each Pi³ PhosphoTyrosine-QE Reagent kit contains material enough for four or eight isolation reactions, respectively.