Document Detail

Optimization of immobilized gallium (III) ion affinity chromatography for selective binding and recovery of phosphopeptides from protein digests.
MedLine Citation:
PMID:  19183793     Owner:  NLM     Status:  MEDLINE    
Although widely used in proteomics research for the selective enrichment of phosphopeptides from protein digests, immobilized metal-ion affinity chromatography (IMAC) often suffers from low specificity and differential recovery of peptides carrying different numbers of phosphate groups. By systematically evaluating and optimizing different loading, washing, and elution conditions, we have developed an efficient and highly selective procedure for the enrichment of phosphopeptides using a commercially available gallium(III)-IMAC column (PhosphoProfile, Sigma). Phosphopeptide enrichment using the reagents supplied with the column is incomplete and biased toward the recovery and/or detection of smaller, singly phosphorylated peptides. In contrast, elution with base (0.4 M ammonium hydroxide) gives efficient and balanced recovery of both singly and multiply phosphorylated peptides, while loading peptides in a strong acidic solution (1% trifluoracetic acid) further increases selectivity toward phosphopeptides, with minimal carryover of nonphosphorylated peptides. 2,5-Dihydroxybenzoic acid, a matrix commonly used when analyzing phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry was also evaluated as an additive in loading and eluting solvents. Elution with 50% acetonitrile containing 20 mg/mL dihydroxybenzoic acid and 1% phosphoric acid gave results similar to those obtained using ammonium hydroxide as the eluent, although the latter showed the highest specificity for phosphorylated peptides.
Uma K Aryal; Douglas J H Olson; Andrew R S Ross
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of biomolecular techniques : JBT     Volume:  19     ISSN:  1943-4731     ISO Abbreviation:  J Biomol Tech     Publication Date:  2008 Dec 
Date Detail:
Created Date:  2009-02-02     Completed Date:  2009-04-02     Revised Date:  2013-06-02    
Medline Journal Info:
Nlm Unique ID:  100888641     Medline TA:  J Biomol Tech     Country:  United States    
Other Details:
Languages:  eng     Pagination:  296-310     Citation Subset:  IM    
National Research Council, Plant Biotechnology Institute, Saskatoon, SK, Canada.
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MeSH Terms
Caseins / chemistry,  isolation & purification
Chromatography, Affinity / methods*
Hydrophobic and Hydrophilic Interactions
Isoelectric Point
Peptide Fragments / chemistry,  isolation & purification
Phosphopeptides / chemistry,  isolation & purification*
Proteomics / methods*
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Reg. No./Substance:
0/Acids; 0/Caseins; 0/Gentisates; 0/Peptide Fragments; 0/Phosphopeptides; 0/Solvents; 7440-55-3/Gallium; VP36V95O3T/2,5-dihydroxybenzoic acid

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