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

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 (HILIC) as a first dimension separation to separate and simplify protein digest mixtures into discrete fractions. Phosphopeptides were then enriched offline using TiO2-coated magnetic beads and subsequently detected online by C18 reverse phase nanoflow HPLC using data-dependent MS/MS High-Energy Collision-activated Dissociation (HCD) fragmentation on a high performance Orbitrap hybrid tandem mass spectrometer. We identified 15533 unique phosphopeptides in 3574 putative phosphoproteins. Systematic computational analysis revealed biological pathways and phosphopeptides 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.