Rapid Combinatorial ERLIC–SCX Solid-Phase Extraction for In-Depth Phosphoproteome Analysis

Protein
phosphorylation is an important mechanism of cellular signaling,
and many proteins are precisely regulated through the interplay of
stimulatory and inhibitory phosphorylation sites. Phosphoproteomics
offers great opportunities to unravel this complex interplay, generating
a mechanistic understanding of vital cellular processes. However,
protein phosphorylation is substoichiometric and, in particular, peptides
carrying multiple phosphorylation sites are extremely difficult to
detect in a highly complex mixture of abundant nonphosphorylated peptides.
Chromatographic methods are employed to reduce sample complexity and
thereby significantly increase the number of phosphopeptide identifications.
We previously demonstrated that combinatorial strong cation exchange–electrostatic
repulsion–hydrophilic interaction chromatography yields a surplus
in overall identifications of phosphopeptides compared with single
chromatographic approaches. Here we present a simple and rapid strategy
implemented as solid-phase extraction not requiring specific instrumentation
such as off-line HPLC systems. It is inexpensive, adaptable for high
and low amounts of starting material, and saves time by allowing multiplexed
sample preparation without any carry-over problem.