Exploring the Human Leukocyte Phosphoproteome Using a Microfluidic Reversed-Phase−TiO2−Reversed-Phase High-Performance Liquid Chromatography Phosphochip Coupled to a Quadrupole Time-of-Flight Mass Spectrometer

摘要

The study of protein phosphorylation events is one of thenmost important challenges in proteome analysis. Despite thenimportance of phosphorylation for many regulatory processesnin cells and many years of phosphoprotein andnphosphopeptide research, the identification and characterizationnof phosphorylation by mass spectrometry is still anchallenging task. Recently, we introduced an approach thatnfacilitates the analysis of phosphopeptides by performingnautomated, online, TiO2 enrichment of phosphopeptidesnprior to mass spectrometry (MS) analysis. The implementationnof that method on a “plug-and-play” microfluidicnhigh-performance liquid chromatography (HPLC)nchip design will potentially open up efficient phosphopeptidenenrichment methods enabling phosphoproteomicsnanalyses by a broader research community. Followingnour initial proof of principle, whereby the device wasncoupled to an ion trap, we now show that this so-callednphosphochip is capable of the enrichment of largennumbers of phosphopeptides from complex cellularnlysates, which can be more readily identified whenncoupled to a higher resolution quadrupole time-of-flightn(Q-TOF) mass spectrometer. We use the phosphochip-nQ-TOF setup to explore the phosphoproteome of nonstimulatednprimary human leukocytes where we identifyn1012 unique phosphopeptides corresponding to 960ndifferent phosphorylation sites providing for the first timenan overview of the phosphoproteome of these importantncirculating white blood cells.