Ultra Acidic Strong Cation Exchange Enabling the Efficient Enrichment of Basic Phosphopeptides

We present a straightforward method to enrich phosphopeptides
with multiple basic residues, an under-represented class in common
enrichment strategies. Our method is based on a two-dimensional strong
cation exchange (SCX) strategy, operating at two different acidic
pHs, enabling both separation and enrichment of different classes
of phosphopeptides. The principle of enrichment is based on the change
of net charge of phosphorylated peptides under strong acidic conditions
in the second SCX, whereas the net charge of regular peptides remains
unchanged, thus enabling separation based on net charge. Application
of our tandem SCX approach to a modest amount of human cells allowed
the identification of over 10 000 unique “basic”
phosphopeptides of which many represent putative targets of basophilic
kinases.