Phosphoprotein affinity resins and methods for making and using the same
View Patent ↗Phosphorylated protein (i.e., phosphoprotein) affinity resins and methods for making and using the same are provided. The subject resins include a substrate bonded to aspartate-based tetradentate ligand/metal ion complexes, where the tetradentate ligand/metal ion complexes have high specificity for phosphorylated amino acids. The subject resins find use in a variety of different applications, including phosphoprotein enrichment applications. Also provided are kits and systems that include the subject resins.
1. A method of making a phosphoprotein affinity resin, said method comprising:
(a) contacting an uncharged aspartate-based resin with a metal ion source comprising metal ions having a 3 + oxidation state and having selective affinity for phosphorylated amino acids to produce a metal ion charged aspartate-based resin, wherein said aspartate-based resin comprises a tetradentate ligand and said contacting occurs in the presence of acetic acid; and
(b) removing non-complexed metal ion from said charged aspartate-based resin to produce said phosphoprotein affinity resin.
2. The method according to claim 1 , wherein said metal ion source comprises Fe 3+ .
3. The method according to claim 1 , wherein said metal ion source comprises lanthanide ion.
4. The method according to claim 3 , wherein said metal ion source comprises Eu 3+ .
5. The method according to claim 1 , wherein said aspartate-based resin is synthesized from an aspartic acid.
6. The method according to claim 5 , wherein said aspartate-based resin is an alkylated aspartate-based resin.
7. The method according to claim 6 , wherein said alkylated aspartate-based resin is a carboxymethylated aspartate-based resin.
8. The method according to claim 1 , wherein said method further comprises contacting said phosphoprotein affinity resin with a dicarboxylic acid to produce a stabilized phosphoprotein affinity resin.
9. The method according to claim 8 , wherein said dicarboxylic acid has the formula:
HOOC—(CH 2 ) n —COOH
wherein:
n is an integer ranging from about 1 to 10.
10. The method according to claim 9 , wherein n is an integer ranging from about 2 to about 4.
11. The method according to claim 8 , wherein said stabilized phosphoprotein affinity resin is combined with an aqueous solution of a lower alcohol to produce a storage stable composition of said stabilized phosphoprotein affinity resin.