Selective enrichment and fractionation of phosphopeptides from peptide mixtures by isoelectric focusing after methyl esterification.
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 alpha-S-casein and beta-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.
Xu
Chong-Feng
CF
Department of Pharmacology and Skirball Institute of Biomolecular Medicine, and Department of Biochemistry, NYU School of Medicine, New York, New York 10016, USA.
Wang
Huaibin
H
Li
Daming
D
Kong
Xiang-Peng
XP
Neubert
Thomas A
TA
D000098:Acetophenones / Q000737:chemistry; D002364:Caseins / Q000737:chemistry; D004951:Esterification; D005841:Gentisates / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D008745:Methylation; D010446:Peptide Fragments / Q000737:chemistry*; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
D000098:Acetophenones; D002364:Caseins; D005841:Gentisates; D010446:Peptide Fragments; D010748:Phosphopeptides; C019339:2,6-dihydroxyacetophenone; D014357:Trypsin; C010925:2,5-dihydroxybenzoic acid