Aging is a biological process that occurs in every living organism at the cellular and molecular level. However, the molecular mechanisms of aging are poorly understood yet. Aging-related protein alterations are implicated in the functional deterioration of cells, tissues, and organs in aged individuals. Proteomic investigations into the agingrelated protein alterations have been thought to be an effective approach to the molecular mechanisms of aging. Replicative aging has been studied as a model of cellular aging in which normal mitotic cells may divide a limited number of times, eventually undergoing a growth arrest termed cellular senescence [1-3]. Protein alterations occurring in the replicative aging were first profiled by 2-D gel-based proteome analysis in the normal human diploid fibroblast line TIG-3 [4]. The replicative aging-related variations of protein expression could be categorized into five patterns as a result of the comparative image analysis of protein spots

on 2-D gel maps. Among them the most interesting variation was the transitional increase at around 60 PDL (population doubling levels), after that the doubling time of the cell population was extended in a logarithmic scale. The spots of stathmin, Hsp60, Hsp70, TCTP, and SOD1 showed such transitional increase suggesting the involvement in the molecular process of replicative aging. The database have been constructed on the aging-related protein alterations, and opened for free access at the URL https://www.proteome.jp/2D/Fibro/humfb_menu.html. In the other aspect, terminally differentiated cells in the post-mitotic state show a distinct phenotype of functional cell aging. Damage accumulated in the cells under oxidative stress is implicated in functional cell aging and in geriatric disorders such as vascular and neurodegenerative diseases. The comprehensive analysis was performed on the aging-related protein alterations in mouse brain tissues by Tsugita et al. [5] first, in which five regions at five ages from the 10th week to the 24th month were compared by 2-D gel electrophoresis. Over 1,000 protein spots were detected by silver staining, and 17 protein spots were found varying in quantity in the course of aging. Among them, the age-dependent decrease was found in histone H3 spot of pI 5.5, suggesting the involvement of the hyper-acetylated form of histone H3 in the mechanism of hippocampal aging. 10.1 IntroductionBiological aging is an inevitable process in which functional deterioration gradually proceeds in almost all tissues and organs throughout the individual life span, especially after maturation. Aging-related alteration appears in both mitotic and post-mitotic cells. Replicative aging is a typical phenotype of cellular aging in which normal mitotic cells count the number of times they have divided, eventually undergoing a growth arrest termed cellular senescence. The reduced mitotic potential of replicative cells in reproductive tissues may cause age-dependent impairment of tissue regeneration. The replicative aging of normal human diploid fibroblasts was first found by Hayflick and Moorhead [1, 2] and recommended as a model of cellular senescence. The shortening of telomere, found

in the process of replicative aging by Harley et al. [3], is the major event counting the number of replication, however, not all the phenotypes of senescent cells, such as reduction in cell migration [6], accumulation of auto-fluorescent proteins and enlargement of cell size, could be ascribed to the shortening of telomere. The human diploid fibroblast strain TIG-3 [7] established in Tokyo Metropolitan Institute of Gerontology has also a finite life span that is defined by the limited number of times the cell can divide, even under an optimal culture condition, unless otherwise immortalized. The replicative aging of TIG-3 was thought to be useful model for comprehensive profiling of aging-related proteome alterations. 2-D PAGE [8, 9] has been widely used in various fields of research for detecting protein alterations in biological processes such as cell differentiation, immortalization and aging. Celis and co-workers [10] established their original 2-D gel protein database of MRC-5 human embryonal lung fibroblast, which was applied to the quantitative profiling of polypeptides of which relative abundance differed between quiescent, proliferating, and SV40-transformed cells [10]. However, no database describing age-related protein alterations have been established yet. Thus, we decided to construct our original 2-D gel-based proteome database upon replicative aging of TIG-3 [4]. The clickable map of protein spots was prepared from silverstained 2-D gel images. The data of relative abundance of proteins were linked to the image map. 10.2 Two-Dimensional Gel Electrophoresis

Two-dimensional (2-D) gel electrophoresis-based quantitative proteome analysis is appropriate for screening of protein alterations in both expression level and posttranslational modification appearing in replicative aging of mitotic cells, and functional aging of post-mitotic cells. In our studies on replicative aging-related proteome, proteins extracted from normal human diploid fibroblast TIG-3 cells at various population doubling levels (PDL), were separated by isoelectric focusing on a strip of immobilized pH gradient (IPG-IEF) in the first dimensional direction, then dissolved by sodium dodecyl

sulfate electrophoresis on a polyacrylamide gel slab (SDS-PAGE) in the second-dimensional direction. Protein spots were stained with silver and quantitated by image processing. Corresponding protein spots were matched across all 2-D gel images, and the profiles in quantitative alteration of each protein spots in the course of cellular aging were categorized into five patterns, (i) simple increase, (ii) simple decrease, (iii) decrease after transient increase, (iv) increase after transient decrease, and (v) irregular or no significant variation. The results of the aging related proteome analysis were stored in our original database, TMIG-2DPAGE, in which the information about the relative abundance of each spots determined by the quantitative 2-D gel image analysis was managed. An alternative “gel-free” method based on LC shotgun MS has been also developed for quantitative proteome analysis [11, 12]. However, major parts of the information about the real protein structure including multiple post-translational modifications have been lost in the process of tryptic digestion. In our preliminary studies, many spots of different pI and molecular mass were assigned to a common gene product by Mascot search, suggesting they are processed by multiple post-translational modifications. This was the reason why we decided to use the 2-D gel-based method for analyzing age-related proteome. 10.2.1 Preparation of Cell Proteins for Analyzing

TIG-3 human fetal lung diploid fibroblasts line established in our institute [7] was employed in the proteomic investigation on replicative aging. The cell line had been confirmed free from mycoplasma contamination. The karyotype of the cell line was analyzed by Matsuo et al. [7]. The cells were serially subcultured in Eagle’s basal medium (BME, GIBCO) supplemented with 10% fetal bovine serum (FBS) by a standard method of 1:4 splitting throughout the in vitro life span. The cell line showed the replicative senescence around 76-80 PDL in the culture condition. Cells at various PDLs were grown to be 70-80% confluent. After rinsing with phosphate-buffered saline, cells were harvested by scraping with a plastic scraper without using trypsin. The cell suspension was transferred to a microfuge tube, and supplemented with urea (8 M), 2-ME (3%), SDS (0.02%) and Triton X-100 (0.1%

in final concentration). Cells were disrupted by ultrasonication, and the supernatant was removed by centrifugation. 10.2.2 Two-Dimensional Gel Electrophoresis

In our studies on replicative aging, 2-D gel electrophoresis was performed mainly according to the ISO-DALT method with slight modifications reported in our previous paper [4]. The original method of high-resolution two-dimensional gel electrophoresis (2-DE), established by O’Farrell [8], is a powerful tool for resolving hundreds of proteins in a crude sample as isolated spots. The spots can be quantified by image analysis and identified by mass spectrometry. In the first dimension, proteins were resolved by isoelectric focusing (IEF) in accordance with their net charge. In the second dimension, the proteins were further separates by SDS-PAGE depending on their molecular mass. An improved method of 2-DE, ISO-DALT, established by Görg [9] was performed in our studies on replicative aging-related proteome, in which isoelectric focusing was carried out on a gel strip of immobilized pH gradient in the first dimension (IPG-IEF). IPG-IEF has a critical advantage over the conventional method of isoelectric focusing in which pH gradient was generated using carrier ampholytes. The un-immobilized pH gradient is not stable and drifts toward the cathodic direction in a long term focusing. After the IPG-IEF gel strip was equilibrated with SDS-containing buffer, and fixed on the top of a slab gel for SDS-PAGE in the second dimension. In the second-dimensional SDS-PAGE, proteins are further separated according to their molecular mass. 10.2.3 Silver Staining and CBB Staining of 2-D Gel

2-D gel-based quantitative proteome research relies on methods for in-gel protein detection. After 2-DE, an appropriate method in visualizing protein spots on a 2-D gel should be carried out for assuring the quantitative analysis. Staining with Coomassie Brilliant Blue (CBB) [13] has been used as the most reliable method for quantitative visualization of protein spots on a 2-D gel. CBB is thought to bind to proteins through electrostatic interactions between the sulfonic groups of the dye and the basic side groups of amino acids [13, 14]. However, the sensitivity of CBB-stain is

not high enough for detecting low abundant protein spots on an analytical 2-D gel in which a limited amount of protein sample could be subjected to the analysis. Silver staining [15] has been developed as an alternative method for visualizing proteins in polyacrylamide gel with the significant advantage in sensitivity over CBB staining. The original methods of silver staining, in which glutaraldehyde was used as cross-linking reagent, could not be applicable to gels for mass-spectrometric protein identification. Mass spectrometry (MS)-compatible silver staining [16] has been developed, in which glutaraldehyde treatment was omitted. However, the MS-compatible silver staining compromises sensitivity in detection. Thus, in our research on replicative aging-related proteome, we decided to use the modified procedure of silver staining [17] with slight modifications for achieving the enhanced uniform sensitivity and acceptable linearity in the dynamic range a standard method of silver staining in which glutaraldehyde-based sensitizes in the fixing and sensitization step was performed to achieve an adequate sensitivity and dynamic range for quantitative proteomics. The 2-D gel, on which higher amount of protein sample was separated for protein identification, was stained with Coomassie Brilliant Blue (CBB).