Proteomic detection of globin gene expression nuclear factors : implementation of an HPLC-CE two-dimensional separation system.

Abstract

Proteomics is one of the fastest growing fields in the life sciences. The current method of choice for studying the proteome of a cell is two-dimensional gel electrophoresis followed by mass spectrometric analysis of tryptic fragments after in-gel digestion of the protein spots. Although this method is used extensively in proteomics, it does exhibit various shortcomings, where nuclear proteins are tough to separate with this system, primarily owing to their low abundance at their natural cellular concentrations. In order to address such shortcomings the focus of proteomics is shifting towards the implementation of a highly sensitive multidimensional high-pressure liquid chromatography - capillary electrophoresis system Yet, to date no documentation regarding nuclear protein separations using this system was submitted. The addition of hydroxyurea to a K562 cell culture induces their differentiation to erythroid-specific cells, with the consequent synthesis heme, indicative of globin chain synthesis. At 96 hours of incubation a maximum number of cells synthesizing heme were observed, as detected by the benzidine-oxidation test. Nuclear extracts from induced and non-induced K562 cells were injected into the ion-exchange HPLC separation unit. This represented the first dimension separation based on the specific isoelectric points of the nuclear proteins, including erythroid Kruppel-like factor (pI 6.71), fetal Kruppel-like factor 1 (pI 8.45) and fetal Kruppel-like factor 2 (pI 9.99). An average of three protein zones, detected as peaks along the elution profile, were eluted from this separation. These protein zones were collected as fractions and injected into a capillary zone electrophoresis system. This second dimension separation yielded a number of protein zones present within the nucleus at the %-hour incubation time. Marked differences in profiles were observed between the non-induced and hydroxyurea-induced K562 cells. The difference in profiles was significant of the "switching on" and "off' of the various genes during the differentiation process resulting in the synthesis of globin chains. The HPLC-CE two-dimensional separation procedure was employed to construct a protein zone map for human erythroid blast-forming-unit (BFU-E) nuclear extracts, which were treated with stem cell factor (another known fetal hemoglobin synthesis inducer in erythroid-lineage cells). Again marked differences were observed between the treated and untreated (control) BFU-E's, indicative of the activation and silencing of genes consequent to differentiation followed by heme synthesis and its incorporation into globin chains. In conclusion, good separations were observed with both the K562 cell nuclear extracts and particularly the human BFU-E nuclear extracts, possibly owing to the use of wide range of protease inhibitors in the nuclear protein extraction procedure. A final identification system, using contemporary mass spectrometry techniques coupled with the HPLC-CE system, can provide the basis for elucidation of the nuclear proteome, thus allowing the targeting of significant proteins in gene expression, as therapeutic drug targets, not only for the hemoglobinopathies described within, but also for the many tissue-specific diseases, such as cancer.