Single-Step Inline Hydroxyapatite Enrichment Facilitates Identification and Quantitation of Phosphopeptides from Mass-Limited Proteomes with MudPIT

Herein we report the characterization and optimization
of single-step
inline enrichment of phosphopeptides directly from small amounts of
whole cell and tissue lysates (100–500 μg) using a hydroxyapatite
(HAP) microcolumn and Multidimensional Protein Identification Technology
(MudPIT). In comparison to a triplicate HILIC-IMAC phosphopeptide
enrichment study, ∼80% of the phosphopeptides identified using
HAP-MudPIT were unique. Similarly, analysis of the consensus phosphorylation
motifs between the two enrichment methods illustrates the complementarity
of calcium- and iron-based enrichment methods and the higher sensitivity
and selectivity of HAP-MudPIT for acidic motifs. We demonstrate how
the identification of more multiply phosphorylated peptides from HAP-MudPIT
can be used to quantify phosphorylation cooperativity. Through optimization
of HAP-MudPIT on a whole cell lysate we routinely achieved identification
and quantification of ca. 1000 phosphopeptides from a ∼1 h
enrichment and 12 h MudPIT analysis on small quantities of material.
Finally, we applied this optimized method to identify phosphorylation
sites from a mass-limited mouse brain region, the amygdala (200–500
μg), identifying up to 4000 phosphopeptides per run.