In-gel tagging and in-gel digestion for phosphoproteins...

Details In-gel tagging and in-gel digestion for phosphoproteins... In-gel tagging and in-gel digestion for phosphoproteins...

2004-10-08

2009-12-08

Carlson, Karen Cochrane (Department: 1657)

Electric lamp and discharge devices

Having vapor generating material

Reexamination Certificate

active

07629744

ABSTRACT:
The present invention relates to a method for phosphorylation site-specific labeling of phosphoproteome with a site-specific tagging reagent and analyzing of the resulting labeled one, more especially, a method for in-situ tagging of phosphorylation sites of phosphoproteins retained in polymeric gel with a nucleophilic tagging reagent. It also relates a method for generating new proteolytic cleavable sites at formerly phosphorylation sites by a proper choice of a nucleophilic tagging reagent. It also relates to a method for phosphopeptides analysis and phosphorylation site identification by in-gel digestion of the previously in-gel tagged proteins and subsequent mass analysis of the resulting peptides. The invention provides in-gel chemical tagging method for phosphoaminoacid residue of phosphoproteins retained in polymeric gel matrix. Phosphoprotein can be immobilized into gel matrix by a variety of methods such as gel electrophoresis. The immobilized phosphoproteins are retained in gel matrix during tagging reaction to phosphorylated aminoacid residue of phosphoproteins, and the resulting tagged proteins are also retained in gel matrix till following purification steps like washing of the tagging reagents are accomplished. The tagged proteins is digested by protease, and the resulting digested peptides is released from gel into solution and applied for peptide mass analysis.

REFERENCES:
patent: 1020050079558 (2005-08-01), None
Oda et al. “Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome”, Nature Biotechnology, 2001, 19:379-382.
Stensballe et al. “Simplified sample preparation method for protein identification by matrix-assisted laser desorption-ionization mass spectrometry: in-gel digestion on the probe surface”, Proteomics, 2001, 1:955-966.

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