Spectral matching sequence database search engines commonly used on mass spectrometry-based proteomics experiments excel at identifying peptide sequence ions, and in addition, possible sequence ions carrying post-translational modifications (PTMs), but most do not provide confidence metrics for the exact localization of those PTMs when several possible sites are available. Localization is absolutely required for downstream molecular cell biology analysis of PTM function in vitro and in vivo. Therefore, we developed PTMProphet, a free and open-source software tool integrated into the Trans-Proteomic Pipeline, which reanalyzes identified spectra from any search engine for which pepXML output is available to provide localization confidence to enable appropriate further characterization of biologic events. Localization of any type of mass modification (e.g., phosphorylation) is supported. PTMProphet applies Bayesian mixture models to compute probabilities for each site/peptide spectrum match where a PTM has been identified. These probabilities can be combined to compute a global false localization rate at any threshold to guide downstream analysis. We describe the PTMProphet tool, its underlying algorithms and demonstrate its performance on ground-truth synthetic peptide reference datasets, one previously published small dataset, one new larger dataset, and also on a previously published phosphoenriched dataset where the correct sites of modification are unknown. Data have been deposited to ProteomeXchange with identifier PXD013210.

Summary. An anatomical analysis of the innervation of murine femora revealed intimate association of haemopoietic and stromal cells with nerve fibres. The mechanical denervation of these femora resulted in significant mobilization of cells into the peripheral blood within 24 h. There was a decrease in femoral cellularity and analysis of the type of cells mobilized also revealed that there was an increase in progenitor cells in the peripheral blood. In non-splenectomized mice these progenitor cells were quickly cleared from the circulation. Chemical sympathectomy of mice with 6-hydroxydopamine resulted in decreased bone marrow cellularity without a change in bone marrow or peripheral blood progenitor cell numbers, nor the sustained rise in peripheral leucocytes observed with whole nerve denervation. These observations argue for selective control of mobilization by the nervous system and also indicate possible control of proliferation within the bone marrow. We conclude that the innervation has an important role in the maintenance of the blood-marrow interface, control of peripheral blood cell numbers, and mobilization of colony forming cells into the periphery.

A number of haematopoietic precursor cell lines have been established which exhibit an absolute dependence on haematopoietic cell growth factor (HCGF) which is secreted by WEHI‐3 myelomonocytic leukaemia cells. In the presence of HCGF, ATP levels are maintained in these factor‐dependent cells (FDC‐P cells); in the absence of HCGF, intracellular ATP levels undergo a steady depletion. The cell death that follows this ATP depletion can be prevented by supplying exogenous ATP suggesting that HCGF maintains these cells via its effects on energy metabolism. We have investigated the effect of HCGF on FDC‐P cells further and found that: (i) HCGF markedly and rapidly increases lactate production; (ii) high extracellular glucose or glycolytic intermediate concentrations can maintain FDC‐P cell viability to some extent whilst stimulating lactate production; (iii) the uptake of 2‐deoxyglucose by FDC‐P2 cells is stimulated by HCGF in a dose‐dependent fashion. This uptake is inhibited by cytochalasin B; (iv) HCGF does not stimulate L‐glucose uptake by FDC‐P cells. These results suggest that HCGF acts to maintain FDC‐P cells via its action on glucose transport. The significance of these results to haemopoiesis and leukaemogenesis is discussed.

Key Points• BCL-2 homology domain 3 mimetic inhibitor ABT-737 targets leukemia initiating cells and progenitors.• Dephosphorylates RAS signaling proteins and regulates proliferation and differentiation genes detected by gene expression profiling.Myelodysplastic syndrome (MDS) transforms into an acute myelogenous leukemia (AML) with associated increased bone marrow (BM) blast infiltration. Using a transgenic mouse model, MRP8[NRASD12/hBCL-2], in which the NRAS:BCL-2 complex at the mitochondria induces MDS progressing to AML with dysplastic features, we studied the therapeutic potential of a BCL-2 homology domain 3 mimetic inhibitor, ABT-737. Treatment significantly extended lifespan, increased survival of lethally irradiated secondary recipients transplanted with cells from treated mice compared with cells from untreated mice, with a reduction of BM blasts, Lin-/Sca-1 1 /c-Kit 1 , and progenitor populations by increased apoptosis of infiltrating blasts of diseased mice assessed in vivo by technicium-labeled annexin V single photon emission computed tomography and ex vivo by annexin V/7-amino actinomycin D flow cytometry, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, caspase 3 cleavage, and re-localization of the NRAS:BCL-2 complex from mitochondria to plasma membrane. Phosphoprotein analysis showed restoration of wild-type (WT) AKT or protein kinase B, extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase patterns in spleen cells after treatment, which showed reduced mitochondrial membrane potential. Exon specific gene expression profiling corroborates the reduction of leukemic cells, with an increase in expression of genes coding for stem cell development and maintenance, myeloid differentiation, and apoptosis. Myelodysplastic features persist underscoring targeting of BCL-2-mediated effects on MDS-AML transformation and survival of leukemic cells. (Blood. 2013; 122(16):2864-2876