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Protein phosphorylation is a critical signal transduction event in many areas of therapeutic interest, including cancer and inflammation. Understanding of these signaling events, their role in disease, or their modulation by therapy, necessitates identification and characterization of phosphoproteins regulated by hormones, circulating factors, or pharmacological agents. Identification of regulated phosphoproteins requires sensitive and robust methods of phosphoprotein enrichment, profiling, identification, and characterization. In this study we utilized a well-characterized cellular inflammation model, LPS-stimulated and drug treated THP-1 cells, to quantitatively assess the enrichment of known and total phosphoproteins with a commercial phosphoprotein enrichment kit. We measured the enrichment of Hsp27 with pan- and phospho-specific antibodies using flourescence based assays and 1D/2D Western blots, and measured total phosphoprotein enrichment with two phosphoprotein quantitation kits. These enriched fractions were then processed and profiled by 2D-DIGE to identify known and unique regulated phosphoproteins. Several phosphoprotein candidates were characterized by 1D- and 2D-Western blotting to verify regulation. Our results indicate that current phosphoprotein enrichment reagents are: 1) subject to poor recovery and preferential enrichment of multiply phosphorylated proteins; 2) that sample processing (desalting) of enriched intact proteins for 2D gel analysis can lead to significant sample loss and artifactual regulation, and 3) that verification of regulation by 1D and 2D Western blotting is an essential step in the identification of phosphoprotein markers of interest.