Proteomic Studies for the Investigation of γ-Globin Induction by Decitabine in Human Primary Erythroid Progenitor Cultures.

Reactivation of γ-globin is considered a promising approach for the treatment of ß-thalassemia and sickle cell disease. Therapeutic induction of γ-globin expression, however, is fraught with lack of suitable therapeutic targets. The aim of this study was to investigate the effects that treatment with decitabine has on the proteome of human primary erythroid cells from healthy and thalassemic volunteers, as a means of identifying new potential pharmacological targets. Decitabine is a known γ-globin inducer, which is not, however, safe enough for clinical use. A proteomic approach utilizing isobaric tags for relative and absolute quantitation (iTRAQ) analysis, in combination with high-pH reverse phase peptide fractionation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), was employed to investigate the effects of decitabine treatment. Bioinformatics analysis making use of the Database for Annotation, Visualization and Integrated Discovery (DAVID) was employed for functional annotation of the 192 differentially expressed proteins identified. The data are available via ProteomeXchange with identifier PXD006889. The proteins fall into various biological pathways, such as the NF-κB signaling pathway, and into many functional categories including regulation of cell proliferation, transcription factor and DNA binding, protein stabilization, chromatin modification and organization, and oxidative stress proteins.

Limitations of current proteomics technologies.

Application of proteomics technologies in the investigation of biological systems creates new possibilities in the elucidation of biopathomechanisms and the discovery of novel drug targets and early disease markers. A proteomic analysis involves protein separation and protein identification as well as characterization of the post-translational modifications. Proteomics has been applied in the investigation of various disorders, like neurological diseases, and the application has resulted in the detection of a large number of differences in the levels and the modifications of proteins between healthy and diseased states. However, the current proteomics technologies are still under development and show certain limitations. In this article, we discuss the major drawbacks and pitfalls of proteomics we have observed in our laboratory and in particular during the application of proteomics technologies in the investigation of the brain.

Proteomic Analysis of Liver from Transgenic Mice Overexpressing Small Heterodimer Partner.

The small heterodimer partner (SHP) is a key regulator of genes involved in cholesterol-bile acid homeostasis and functions as a specific transcription repressor. Differential protein expression in the liver of transgenic mice expressing the human SHP gene was compared with wild-type animals. Liver protein extracts were analyzed by two-dimensional electrophoresis and the proteins were identified by MALDI-TOF-MS. Approximately 30 proteins were differentially-expressed in the livers of transgenic mice, compared to the control mice. Major effects were evident in lipid accumulation, including a fatty acid-binding protein. Overexpression of SHP also triggered alterations in key enzymes involved in the metabolism of amino acids, nucleic acids and urea and was associated with changes in cellular proteins involved in calcium homeostasis, detoxification and protein folding and repair.