Quantitative proteomics analysis of phosphorylated proteins in the hippocampus of Alzheimer's disease subjects

Abstract

Phosphorylation on tyrosine, threonine and serine residues represents one of the most important post-translational modifications and is a key regulator of cellular signaling of multiple biological processes that require a strict control by protein kinases and protein phosphatases. Abnormal protein phosphorylation has been associated with several human diseases including Alzheimer's disease (AD). One of the characteristic hallmarks of AD is the presence of neurofibrillary tangles, composed of microtubule-associated, abnormally hyperphosphorylated tau protein. However, several others proteins showed altered phosphorylation levels in AD suggesting that deregulated phosphorylation may contribute to AD pathogenesis. Phosphoproteomics has recently gained attention as a valuable approach to analyze protein phosphorylation, both in a quantitative and a qualitative way. We used the fluorescent phosphospecific Pro-Q Diamond dye to identify proteins that showed alterations in their overall phosphorylation in the hippocampus of AD vs. control (CTR) subjects. Significant changes were found for 17 proteins involved in crucial neuronal process such as energy metabolism or signal transduction. These phosphoproteome data may provide new clues to better understand molecular pathways that are deregulated in the pathogenesis and progression of AD.

Figure 1

Representative 2D phosphorylation maps of AD (top) and CTR (bottom) hippocampus. Gels were stained using Pro-Q Diamond fluorescent dye. The spots showing altered phosphorylation levels between AD and control are labeled. Relative change in phosphorylation, after normalization of the immunostaining intensities to the protein content, was significant for 17 spots.

Figure 2

Representative 2D protein expression maps of AD (top) and CTR (bottom) hippocampus. Gels were stained using Sypro Ruby fluorescent dye. The spots showing altered phosphorylation levels between AD and control are labeled.

Figure 3

Representative enlarged images of selected spots from CTR and AD hippocampus showing the altered phosphorylation of two selected proteins: GAPDH and tER ATPase. In the figure are shown the position of the respective protein spot in the gel in the 2D image, 3D density graphs elaborated by PD-Quest software, and the histograms with phosphorylation (green and red) and expression (blue) intensity bars of each of the 6 samples per group.

Figure 4

Representative scheme of pathologic implications of aberrant phosphorylation in AD.