Selective Enrichment and Fractionation of Phosphopeptides from Peptide Mixtures by Isoelectric Focusing after Methyl Esterification

Abstract

SummaryWe have developed a new strategy to enrich and fractionate phosphopeptides from peptide mixtures based on difference in their isoelectric points (pIs) after methyl esterification. After isoelectric focusing (IEF) of a methylated tryptic digest of a mixture of α-S-casein and β-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 (IPG). 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 based on 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-dihydroxyacetophenone (DHAP) is superior to 2, 5-dihydroxybenzoic acid (DHB) as a matrix for MALDI Q-TOF MS of methylated phosphopeptides in both positive and negative ion modes.

Strong cation exchange (SCX) based analytical methods for the targeted analysis of protein post-translational modifications

“…However, although IEF separates peptides on pI, the pI of phosphorylated and/or N-acetylated peptides can vary over a wide range, limiting the ability of IEF for selective enrichment of such modified peptides. Combining IEF with peptide methyl esterification can lead to enrichment of phosphopeptides [54].…”

Phosphopeptide enrichment using microscale titanium dioxide solid phase extraction

“…Various methods have been used for a selective phosphopeptide enrichment, including strong cationexchange chromatography (SCX) [5,6], IEF [7,8], immobilized metal affinity chromatography (IMAC) [9,10], and metal oxide affinity chromatography (MOAC) [11 -16].…”

Enhanced MALDI-TOF MS Analysis of Phosphopeptides Using an Optimized DHAP/DAHC Matrix

“…It was clear that all phosphopeptides could be easily detected with a low DHAP/DAHC matrix ratio in the range of 1 : 30 to 1 : 60. In an optimal matrix solution, the concentration of DHAP was 1.6–3.3 mg/mL, which is lower than the concentration of 10–15 mg/mL used by other groups [24, 26, 27]. Once the DHAP/DAHC ratio was down to 1 : 80, most multiple phosphorylated peptides could no longer be detected, likely because there were not sufficient numbers of DHAP molecules to transfer the laser energy to the peptides to be protonated.…”