The free-radical theory predicts that the oxidative stress accelerates the rate of aging and increases the onset of degenerative disorders in the elderly. Dopaminergic neurons are especially vulnerable to age-related neuronal disorders due to reactive oxygen species generated in the pathway of dopamine metabolism. Biochemical changes occurring in substantia nigra of Parkinson's disease patients suggest that the oxidative-stress-induced cell damages may be involved in the neurodegeneration. In our previous researches, we found that the dephosphorylation of elongation factor-2 and phosphorylation of nuclear lamin A/C might be neuronal cell specific response to oxidative stress. (Nakamura et al. BBA, 1763(9), 977-989, 2006)
The dephosphorylation and phosphorylation of those proteins are significant biomarkers for analyzing the molecular mechanisms of the stress response, however, such a phosphoproteome analysis is thought to be inappropriate for clinical investigation of neurodegeneration if it was not detectable in cerebrospinal fluid or serum of patients. Thus, we proceeded to the 2D-DIGE analysis of secretome, proteome of secreted proteins, using the culture system in which oxidative stress was applied to human SH-SY5Y neuroblastoma cells. As the result of our secretome analysis, we identified ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2 N, ubiquitin C-terminal hydrorase-L1, 14-3-3 protein isoforms, Rab GDP dissociation inhibitor β, Rho GDP-dissociation inhibitor 1, peroxiredoxin-2, glutathione S-transferase P, α enolase, LDH B chain as oxidative-stress-reporting biomarker candidates.

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The identity of low-molecular-weight and minor protein spots, appeared in 2-DE patterns of human plasma, was examined. They were not obvious in the patterns of “Type-I” 2-DE (non-denaturing IEF followed by non-denaturing gel electrophoresis), but clearly detected in the patterns of “Type II” 2-DE (non-denaturing IEF followed by SDS gel electrophoresis) at pI 5.5-7.5 and apparent mass 8-40 kDa¹⁾. The spots were not obviously detected when the IEF gels were kept at low temperature (around 4°C) during electrophoresis, suggesting that they are the proteolysis products of plasma proteins. The minor spots were more obviously detected when human plasma was subjected to ammonium sulfate (AS) fractionation and the 0-35% saturated AS fraction was dialyzed and subjected to Type-II 2-DE. Then the 116 spots on the 2-DE pattern, detected at pI 5-7.5 and apparent mass 8-60 kDa, were excised and subjected to MALDI-MS measurements and the mass spectra were analyzed using the software of peptide mass fingerprinting (PMF) Mascot and ProFound to assign the proteins. Many of the spots were assigned to contain fibrinogen α chain, especially those at pI 5.5-7.5 and apparent mass 8-40 kDa, suggesting that these spots are its fragments. The distribution of the MS-detected peptide fragments suggested that the molecular-mass heterogeneity might be caused by the cleavage of multiple sites on the α chain. Care must be taken to keep the temperature of IEF gels at around 4°C during electrophoresis, when human plasma proteins are subjected to non-denaturing IEF. The absence of the spots of fibrinogen fragments on Type-II 2-DE gels would validate the intactness of plasma proteins. The advantages of micro gel system for the analysis of intact protein mixtures are suggested.

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