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.