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

Proteomics. 2013 Apr;13(8):1325-33. doi: 10.1002/pmic.201200369. Epub 2013 Mar 6.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD34 / metabolism*
  • Binding Sites
  • CD3 Complex / metabolism
  • Chromatography, Liquid / methods
  • Hematopoietic Stem Cells / metabolism*
  • High-Throughput Screening Assays / methods
  • Humans
  • Magnetics
  • Phosphoproteins / analysis*
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Proline / metabolism
  • Protein Kinases / metabolism
  • Proteomics / methods*
  • Signal Transduction
  • T-Lymphocytes / metabolism
  • Tandem Mass Spectrometry / instrumentation
  • Tandem Mass Spectrometry / methods
  • Titanium / chemistry*
  • Transcription Factors / metabolism


  • Antigens, CD34
  • CD3 Complex
  • Phosphoproteins
  • Transcription Factors
  • titanium dioxide
  • Proline
  • Titanium
  • Protein Kinases