title: 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.
abstract: 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.
authors: Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
authors: Hongbo
authors: H
authors: Guo
authors: Ruth
authors: R
authors: Isserlin
authors: Xiaoji
authors: X
authors: Chen
authors: Weijia
authors: W
authors: Wang
authors: Sadhna
authors: S
authors: Phanse
authors: Peter W
authors: PW
authors: Zandstra
authors: Patrick J
authors: PJ
authors: Paddison
authors: Andrew
authors: A
authors: Emili
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism*; D001665:Binding Sites; D017252:CD3 Complex / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008280:Magnetics; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism*; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D014025:Titanium / Q000737:chemistry*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D017252:CD3 Complex; D010750:Phosphoproteins; D014157:Transcription Factors; C009495:titanium dioxide; D011392:Proline; D014025:Titanium; D011494:Protein Kinases
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