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Integrative network analysis of signaling in human CD34 + hematopoietic progenitor cells by global phosphoproteomic profiling using TiO 2 enrichment combined with 2D LC‐MS/MS and pathway mapping
Author(s) -
Guo Hongbo,
Isserlin Ruth,
Chen Xiaoji,
Wang Weijia,
Phanse Sadhna,
Zandstra Peter W.,
Paddison Patrick J.,
Emili Andrew
Publication year - 2013
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201200369
Subject(s) - phosphopeptide , tandem mass spectrometry , cd34 , phosphoproteomics , chemistry , haematopoiesis , progenitor cell , microbiology and biotechnology , biology , computational biology , mass spectrometry , stem cell , kinase , biochemistry , protein kinase a , chromatography , protein phosphorylation
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 TiO 2 ‐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.