Proteogenomics of the human hippocampus: The road ahead.

The hippocampus is one of the most essential components of the human brain and plays an important role in learning and memory. The hippocampus has drawn great attention from scientists and clinicians due to its clinical importance in diseases such as Alzheimer's disease (AD), non-AD dementia, and epilepsy. Understanding the function of the hippocampus and related disease mechanisms requires comprehensive knowledge of the orchestration of the genome, epigenome, transcriptome, proteome, and post-translational modifications (PTMs) of proteins. The past decade has seen remarkable advances in the high-throughput sequencing techniques that are collectively called next generation sequencing (NGS). NGS enables the precise analysis of gene expression profiles in cells and tissues, allowing powerful and more feasible integration of expression data from the gene level to the protein level, even allowing "-omic" level assessment of PTMs. In addition, improved bioinformatics algorithms coupled with NGS technology are finally opening a new era for scientists to discover previously unidentified and elusive proteins. In the present review, we will focus mainly on the proteomics of the human hippocampus with an emphasis on the integrated analysis of genomics, epigenomics, transcriptomics, and proteomics. Finally, we will discuss our perspectives on the potential and future of proteomics in the field of hippocampal biology. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.

Large-scale analysis of posttranslational modifications in the hippocampus of patients with Alzheimer's disease using pI shift and label-free quantification without enrichment.

Posttranslational modifications modulate protein function in cells. Global analysis of multiple posttranslational modifications can provide insight into physiology and disease, but presents formidable challenges. In the present study, we used a technique that does not require target enrichment to analyze alterations in the phosphorylation and ubiquitination of proteins from patients with Alzheimer's disease (AD). Guided by our previous findings, we applied three strategies to further our understanding of the dysregulation of posttranslationally modified proteins. We first identified phosphorylation sites by determining peptide pI shifts using OFFGEL. Second, using tandem mass spectrometry, we determined the ubiquitination status of the proteins using an assay for a trypsin digestion remnant of ubiquitination (Gly-Gly). Third, for large-scale discovery, we quantified the global differences in protein expression. Of the proteins expressed in AD tissue at levels of 2.0 or greater compared with controls, 60 were phosphorylated and 56 were ubiquitinated. Of the proteins expressed at levels of 0.5 or lower compared with controls, 81 were phosphorylated and 56 were ubiquitinated. Approximately 98 % of the phosphopeptides exhibited a pI shift. We identified 112 new phosphorylation sites (51.38 %), and 92 new ubiquitination sites (96.84 %). Taken together, our findings suggest that analysis of the alterations in posttranslationally modified proteins may contribute to understanding the pathogenesis of AD and other diseases.

Profiling and semiquantitative analysis of the cell surface proteome in human mesenchymal stem cells.

Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for the development of cell therapeutic strategies. MSC surface protein profiles provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets. Basic fibroblast growth factor (bFGF) affects cell surface proteins, which are associated with increased growth rate, differentiation potential, as well as morphological changes of MSCs in vitro. Cell surface proteins were isolated using a biotinylation-mediated method and identified using a combination of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were cut into 20 bands and digested with trypsin. Each tryptic fragment was analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Proteins were identified using the Mascot search program and the International Protein Index human database. Noble MSC surface proteins (n = 1,001) were identified from cells cultured either with (n = 857) or without (n = 667) bFGF-containing medium in three independent experiments. The proteins were classified using FatiGO to elucidate their function. We also confirmed the proteomics results using Western blotting and immunofluorescence microscopic analysis. The nature of the proteins identified makes it clear that MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter proteins, four Ras-related Rab proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are implicated in bFGF-induced signal transduction of MSCs. Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.

Proteome analysis of human cerebrospinal fluid as a diagnostic biomarker in patients with meningioma.

BACKGROUND: To identify meningioma-specific proteins, cerebrospinal fluid (CSF) from 4 patients with a meningioma and 4 patients with a non-brain tumorous lesion were analyzed. MATERIAL/METHODS: Two-dimensional electrophoresis and electrospray quadrupole time-of-flight tandem mass spectrometry analyses revealed 10 unique spots, containing 11 independent proteins (spot #2 and #4 each contained 2 proteins and spot #3 was not identified) were evident in CSF associated with human meningioma: serum albumin precursor (3 different isoforms), Apolipoprotein E (Apo E), Apolipoprotein J precursor (Apo J), Transthyretin precursor (TTR), Prostaglandin D2 synthase 21 kDa (PTGDS), proapolipoprotein, Chain D hemoglobin Ypsilanti, alpha-1-antitrypsin (AAT), and beta-2-microglobulin precursor (ß2M). RESULTS: The contents of Apo E, Apo J and AAT were increased, while PTGDS, TTR and ß2M were decreased. CONCLUSIONS: The results observed by 2-dimensional electrophoresis were verified by Western blot analysis. The unique proteins may represent possible candidate biomarkers of meningioma.