Proteomic study on X-irradiation-responsive proteins and ageing: search for responsible proteins for radiation adaptive response.

We investigated high- or low-dose irradiation-responsive proteins using proteomics on two-dimensional (2D) PAGE, and the effects of ageing on cell responses to radiation in variously aged rat astrocytes. After 5 Gy irradiation, the relative abundance of peroxiredoxin 2, an antioxidant enzyme, and latexin, an inhibitor of carboxypeptidase, increased. The induction of these proteins was suppressed by ageing, suggesting that the response to high-dose radiation decreased with ageing. The relative abundance of elongation factor 2 (EF-2) fragment increased 3 h and reduced 24 h after 0.1 Gy irradiation. Temporal enhancement of the EF-2 fragment due to low-dose irradiation was suppressed by ageing. Since radiation adaptive response in cultured astrocytes was observed 3 h but not 24 h after 0.1 Gy irradiation and suppressed by ageing as previously reported, alteration of the EF-2 fragment corresponded to the radiation adaptive response. We also examined phospho-protein profiles, resulting in the relative abundance of phospho-EF-1beta and phospho-beta-actin being altered by 0.1 Gy irradiation; however, ageing did not affect the alteration of phospho-EF-1beta and phospho-beta-actin, unlike the EF-2 fragment. The results suggested that the EF-2 fragment was a possible candidate for the protein responsible for the radiation adaptive response in cultured astrocytes.

Phosphoproteomic profiling of human SH-SY5Y neuroblastoma cells during response to 6-hydroxydopamine-induced oxidative stress.

Dopaminergic neurons are known to be vulnerable to age-related neuronal disorders due to reactive oxygen species (ROS) generated during dopamine metabolism. However, it remains unclear what kinds of proteins are involved in the response to oxidative stress. We examined changes in whole proteins and phosphoproteins in the human dopaminergic neuroblastoma cell line SH-SY5Y under oxidative stress induced by the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Proteins of SH-SY5Y cells at various stages of oxidative stress were separated by two-dimensional gel electrophoresis for comparative analysis. Increase in glutathione-S-transferase pi was detected on SYPRO Ruby-stained gels by computer-aided image analysis. Stress-induced alterations in phosphoproteins were detected by Pro-Q Diamond staining. Elongation factor 2, lamin A/C, T-complex protein 1, and heterogeneous nuclear ribonucleoprotein H3 were identified by MALDI-TOF mass spectrometry as stress-responsive elements.

Chromatin-related proteins in pluripotent mouse embryonic stem cells are downregulated after removal of leukemia inhibitory factor.

Embryonic stem (ES) cells have generated enormous interest due to their capacity to self-renew and the potential for growing many different cell types in vitro. Leukemia inhibitory factor (LIF), bone morphogenetic proteins, octamer-binding protein 3 or 4, and Nanog are important factors in the maintenance of pluripotency in mouse ES cells. However, the mechanisms by which these factors regulate the pluripotency remain poorly understood. To identify other proteins involved in this process, we did a proteomic analysis of mouse ES cells that were cultured in the presence or absence of LIF. More than 100 proteins were found to be involved specifically in either the differentiation process or the maintenance of undifferentiated state. Among these, chromatin-related proteins were identified as the major proteins in nuclear extracts of undifferentiated cells. Analysis with real-time RT-PCR revealed that enrichment of these proteins in pluripotent ES cells was regulated at the transcriptional levels. These results suggest that specific chromatin-related proteins may be involved in maintaining the unique properties of pluripotent ES cells.

Performance of electrokinetic supercharging for high-sensitivity detection of DNA fragments in chip gel electrophoresis.

Chip gel electrophoresis was explored for high-sensitivity detection of DNA by combining electrokinetic injection with transient isotachophoresis preconcentration (here named electrokinetic supercharging (EKS)). Low concentrations (0.2 mg/L) of DNA sample could be detected without fluorescence labeling using a conventional UV detector (at 260 nm). On a single-channel microchip, identification of PCR product was performed by exploiting both external and internal calibration methods. The deviation between the two calibration methods was about 3.6%, and the identified DNA fragment size matched with the predicted size of the template DNA. On the cross microchip the EKS preconcentration has also been achieved when changing the injection reservoir differing from the one used previously. The procedure was computer-simulated and the influence of the voltage applied to two-side reservoirs on sample preconcentration and dilution was also discussed.

Electrokinetic supercharging preconcentration and microchip gel electrophoretic separation of sodium dodecyl sulfate-protein complexes.

A microchip gel electrophoresis (MCGE) method with electrokinetic supercharging (EKS, electrokinetic injection with transient isotachophoresis) on a single channel chip was developed for high-sensitive detection of a standard mixture of six proteins (phosphorylase b, albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, and alpha-lactalbumin) in the form of sodium dodecyl sulfate (SDS) complexes. An average lower limit of detectable concentration (LLDC) achieved using UV detection at 214 nm was 0.27 microg/mL that is 30 times lower than that of conventional MCGE on a cross geometry chip. The calibration curves for molecular weight and concentration of SDS-protein complexes suggested that the present EKS-MCGE method had a better linear dynamic range and benefited future applications for qualitative and quantitative analysis of unknown protein samples. It was found that an excessive amount of unbound SDS in the sample deteriorated the preconcentration effect and resolution. The developed method appears greatly promising for high-speed and high sensitive analysis of SDS-proteins by MCGE.