Depletion of Acidic Phosphopeptides by SAX To Improve the Coverage for the Detection of Basophilic Kinase Substrates

The Ser/Thr protein kinases fall into three major subgroups,
pro-directed,
basophilic, and acidophilic, on the basis of the types of substrate
sequences that they preferred. Despite many phosphoproteomics efforts
that have been taken for global profiling of phosphopeptides, methodologies
focusing on analyzing a particular type of kinase substrates have
seldom been reported. Selective enrichment of phosphopeptides from
basophilic kinase substrates is difficult because basophilic motifs
are cleaved by trypsin during digestion. In this study, we develop
a negative enrichment strategy to enhance the identification of basophilic
kinase substrates. This method is based on an observation that high
pH strong anion exchange (SAX) chromatography can separate tryptic
phosphopeptides according to the number of acidic amino acidic residues
that they have. Thus, SAX was applied to deplete acidic phosphopeptides
from the phosphopeptide mixture, which improved the coverage for the
detection of basophilic kinase substrates. The SAX depletion approach
was further combined with online SCX-RP separation for large-scale
analysis of mouse liver phosphoproteome, which resulted in the identification
of 6944 phosphorylated sites. It was found that motifs associated
with basophilic kinases prevail for these identified phosphorylated
sites.