Integrative network analysis of signaling in human CD34+ hematopoietic progenitor cells by global phosphoproteomic profiling using TiO2 enrichment combined with 2D LC-MS/MS and pathway mapping


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Correspondence: Professor Andrew Emili, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, ON, Canada M5S3E1


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Protein kinase signaling regulates human hematopoietic stem/progenitor cell (HSPC) fate, yet little is known about critical pathway substrates. To address this, we have developed and applied a large-scale, empirically optimized phosphopeptide affinity enrichment strategy with high-throughput 2D LC-MS/MS screening to evaluate the phosphoproteome of an isolated human CD34+ HSPC population. We first used hydrophilic interaction chromatography as a first dimension separation to separate and simplify protein digest mixtures into discrete fractions. Phosphopeptides were then enriched off-line using TiO2-coated magnetic beads and subsequently detected online by C18 RP nanoflow HPLC using data-dependent MS/MS high-energy collision-activated dissociation fragmentation on a high-performance Orbitrap hybrid tandem mass spectrometer. We identified 15 533 unique phosphopeptides in 3574 putative phosphoproteins. Systematic computational analysis revealed biological pathways and phosphopeptide motifs enriched in CD34+ HSPC that are markedly different from those observed in an analogous parallel analysis of isolated human T cells, pointing to the possible involvement of specific kinase-substrate relationships within activated cascades driving hematopoietic renewal, commitment, and differentiation.