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. Issue 8 (6th March 2013)

• Record Type: Journal Article
• Authors: Guo, Hongbo; Isserlin, Ruth; Chen, Xiaoji; Wang, Weijia; Phanse, Sadhna; Zandstra, Peter W.; Paddison, Patrick J.; Emili, Andrew; Figeys, Daniel
• Is Part Of: Proteomics. Volume 13:Issue 8(2013:Apr.)
• Page Start: 1325
• Page End: 1333
• Publication Date: 2013-03-06
• Subjects: Proteins -- Separation -- Periodicals; Bioinformatics -- Periodicals; Proteomics -- Periodicals; Genomes -- Periodicals; Molecular genetics -- Periodicals; 572.605
• DOI: 10.1002/pmic.201200369
• Languages: English
• ISSNs: 1615-9853

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <p>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.</p> </abstract>