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We tested, characterized, and optimized single-step inline enrichment of phosphopeptides directly from whole cell and tissue lysates using a hydroxyapatite (HAP) microcolumn and Multi-dimensional Protein Identification Technology (MudPIT). We applied this optimized method to identify phosphorylation sites from mass-limited samples including a mouse brain region, the amygdala (200 – 500 μg), identifying up to 4000 phosphopeptides per run.
