Web Release Date: January 24,
Selective Enrichment and Fractionation of Phosphopeptides from Peptide Mixtures by Isoelectric Focusing after Methyl Esterification
and
Department of Pharmacology and Skirball Institute of Biomolecular Medicine, and Department of Biochemistry, NYU School of Medicine, New York, New York 10016, and LITEC Systems Corporation, New York, New York 10006
Received for review August 28, 2006. Accepted December 18, 2006.
Abstract:
We have developed a new strategy to enrich and fractionate phosphopeptides from peptide mixtures based on the
difference in their isoelectric points (pIs) after methyl
esterification. After isoelectric focusing (IEF) of a methylated tryptic digest of a mixture of 
-S-casein and 
-casein,
phosphopeptides were selectively enriched at acidic and
neutral pHs while nonphosphopeptides left the focusing
gel because their pIs are higher than the upper limit of
the immobilized pH gradient. We wrote a web-based
program, pIMethylation, to predict the pIs for peptides
with and without methyl esterification. Theoretical calculations using pIMethylation indicated that methylated
phosphopeptides and non-phosphopeptides can be grouped
on the basis of the number of phosphate groups and basic
residues in each peptide. Our IEF results were consistent
with theoretical pIs of methylated peptides calculated by
pIMethylation. We also showed that 2,6-dihydroxy-acetophenone is superior to 2,5-dihydroxybenzoic acid as
a matrix for MALDI Q-TOF MS of methylated phosphopeptides in both positive and negative ion modes.
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