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 Orbit

SUBMITTER: Guo H 

PROVIDER: S-EPMC3883140 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature