T. Toda / Experimental Gerontology 35 (2000) 803±810

Experimental Gerontology 35 (2000) 803±810

803

www.elsevier.nl/locate/expgero

Current status and perspectives of proteomics in
aging research
T. Toda*
Department of Gene Regulation and Protein Function, Tokyo Metropolitan Institute of Gerontology,
35-2 Sakaecho, Itabashi-Ku, Tokyo 173-0015, Japan
Received 3 May 2000; accepted 6 June 2000

Abstract
The accumulation of non-enzymatic modi®cations on both DNA and protein molecules under the
attack of reactive oxygen species (ROS), is one of the most possible factors responsible for the
functional deterioration in aged cells. Direct protein modi®cations as well as DNA damages may be
detectable, in part, by proteome analysis if the gene expression is affected by the damages on DNA.
The novel term ªproteomeº, which is a compound of ªproteinº and ªgenomeº, means a whole set of
proteins expressed in a tissue or a cell strain to be investigated. Proteomics is a methodology for
analyzing proteomes. In proteomics, two-dimensional gel electrophoresis is performed primarily to
separate constitutive proteins, followed by mass spectrometry to identify each protein of interest and
to determine a possible post-translational modi®cation. Proteomics has offered us an innovative tool
for investigating the molecular mechanisms of cellular aging. q 2000 Elsevier Science Inc. All
rights reserved.
Keywords: Proteome; Two-dimensional gel electrophoresis; Mass spectrometry; Protein oxidation

1. Introduction
The age-dependent deterioration of cell function appears in both replicative and postmitotic cells, in living organisms. The major function of stem cells is cell division in the
regeneration of cell population and tissues. Most replicative blast cells play another role:
in the excretion of cytokines for inter-cellular signal transduction. Post-mitotic cells in the
brain, heart, kidney and many other organs have tissue-speci®c functions. Although a
downstream of the cascade in cellular aging may be cell-type speci®c, and may proceed
in a given intracellular environment that is made with all constituents of the proteome,
* Tel. 181-3-39643241; fax: 181-3-35794776.
E-mail address: ttoda@tmig.or.jp (T. Toda).
0531-5565/00/$ - see front matter q 2000 Elsevier Science Inc. All rights reserved.
PII: S 0531-556 5(00)00139-X

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T. Toda / Experimental Gerontology 35 (2000) 803±810

Fig. 1. Possible causes of aging in the mechanisms of life, and the target of ªGerontology Proteomicsº. Cascade of
cellular aging may proceed in an intracellular environment that is made with cell-type-speci®c ªproteomeº. Many
primary causes of cellular aging are commonly speculated for all cell types including mitotic and post-mitotic
cells. Genetic background is one of the most important factor that determine the quality of the defense system
against environmental attacks: Cosmic rays, UV light, reactive oxygen species (ROS), and reducing sugars are
signi®cant elements of environmental attacks. Results of the battle against these environmental attacks may be
revealed in their proteome pro®les.

there may be common factors in the upstream of the cascade. The accumulation of
molecular damages on DNAs and proteins under environmental attacks including oxidative stress, may be one of the major events that trigger the cascade of cellular aging as
described in Fig. 1.
Oxidative protein modi®cation may alter methionine residue to sulfoxide, phenylalanine to diphenyl, and lysyl residue to carbonyl (Smith et al., 1997; Wells Knecht et al.,
1997; Stadtman and Berlett, 1998). In proteomics, such protein altered by post-translational modi®cation, may be separated from the original form by high resolution twodimensional gel electrophoresis (2-DE), and the modi®cation may be determined by
mass spectrometry (MS).
2. Materials and methods
Immobiline DryStrip and Pharmalyte were purchased from Amersham Pharmacia
Biotech KK (Tokyo, Japan). Sequi-Blot PVDF membrane was obtained from the Nippon
Bio-Rad Laboratories (Tokyo, Japan). Acrylamide, N,N H -methylenebisacrylamide and
TEMED were from Daiichi Pure Chemicals (Tokyo, Japan). Sequencing grade endoproteinase Lys-C was from Roche Molecular Biochemicals (Indianapolis, IN, USA). Trizma
base, Tricine, SDS, Triton X-100, iodoacetamide, acetonitrile, tri¯uoroacetic acid and
alpha-cyano 4-hydroxy-cinnamic acid were from Sigma (St. Louis, MO, USA). Quick

T. Toda / Experimental Gerontology 35 (2000) 803±810

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CBB staining reagent, silver staining reagent kit ªWakoº, urea, and other chemicals of
reagent grade were obtained from Wako Pure chemicals (Osaka, Japan).
2.1. Two-dimensional gel electrophoresis
Constitutive proteins in a cell extract were separated into isolated spots by 2-DE
in an ªimmobilized pH gradient isoelectric focusing (IPG-IEF)/sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) systemº according to the
standard protocol (Toda and Kimura, 1997), with minor modi®cations. The details
of the modi®ed protocol are shown in our web homepage: http://proteome.tmig.or.jp/
2D/2DE_method.html. In brief, the ®rst-dimensional IPG-IEF was carried out on a
re-swollen Immobiline DryStrip, pH 4±8, 18-cm long (Code No. 18-1004-34) in the
CoolPhoreStar IPG-IEF Type P horizontal IEF apparatus (Anatech, Tokyo, Japan).
A 20-ml aliquot of the sample solution, which was absorbed in a small piece of ®lter
paper, was applied near the cathode wick on the IPG gel. Spot proteins on a gel
plate for ªdifferential protein displayº were subsequently visualized by silver staining. For the preparative use of 2-DE gel, the IPG gel was swollen in a solution
containing 0.2 ml of cell extract. After 46,700-Vh electrofocusing, the IPG gel was
equilibrated with the SDS-treatment solution (50 mM Tris±HCl, pH 6.8, 6 M urea,
0.5% (w/v) dithiothreitol, 2% (w/v) SDS, 0.005% (w/v) BPB, 25% (v/v) glycerol),
initially for 30 min, and then followed by carbamoylmethylation in an iodoacetamide-containing buffer (50 mM Tris±HCl, pH 6.8, 6 M urea, 4.5% (w/v) iodoacetamide, 2% (w/v) SDS, 0.005% (w/v) bromophenol blue (BPB), 25% (v/v) glycerol)
for 10 min. An equilibrated gel strip was placed on top of a gel slab (7.5%T, 3%C,
20 £ 18 cm†; and ®rmly contacted to the top of a gel slab by pressing the gel strip
with a shark-teeth comb. SDS-PAGE was run vertically in the CoolPhoreStar SDSPAGE Tetra-200 vertical slab gel electrophoresis apparatus (Anatech, Tokyo, Japan)
using a tricine buffer system.
2.2. Protein staining and image processing
Protein spots were visualized on the gel slab for a differential protein display analysis by
silver staining, using the original version of the reagent kit ªWakoº as it showed the widest
dynamic range in optical density among all the commercially available reagents tested.
The 2-DE gel images were acquired using a Sharp JX-330 scanner. Noise reduction,
background subtraction, spot detection, spot quanti®cation, gel-to-gel matching and differential protein analysis were carried out using a PDQuest software (Bio-Rad Laboratories,
Hercules, CA, USA).
2.3. Endoproteinase digestion and peptide mass ®nger printing
After scanning, Coomassie-stained proteins in the gel slab were re-solubilized and
transferred onto a PVDF membrane as follows. The stained gel was rinsed in pure
water thrice, each time for 30 min, incubated in a re-solubilization buffer (0.2% (w/v)
SDS, 0.3% (w/v) Tris, 0.7% (w/v) glycine) for 10 min and mounted on an electrotransfer

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Fig. 2. Detection of age-related protein alteration by 2D gel electrophoresis and quantitation of relative intensity
by image processing in proteome analysis. The relative intensity of ssp7001 shows a transitional increase around
65 PDL, which is the border between phases 2 and 3 of cellular aging of normal human diploid ®broblasts TIG-3.

blotting chamber. The electrotransfer was carried out overnight at 4 V/cm in 0.1% (w/v)
SDS, 0.3% (w/v) Tris and 1.5% (w/v) glycine.
Protein spots were excised from the PVDF membrane and the Coomassie dye was
removed by rinsing in 60% (v/v) methanol. For the MS analysis, the piece of
membrane was incubated with 4.5% (w/v) polyvinyl pyrolidone 25 in 2.5 mM
Tris±HCl, pH 7.7, for 30 min, followed by a brief rinsing in 5% (v/v) acetonitrile.
The digestion was carried out overnight with 0.1 mg of sequencing grade endoproteinase Lys-C in 30 ml of 8% (v/v) acetonitrile, 2.5 mM Tris±HCl, pH 7.7, at 378C.
After digestion, the reaction mixture was supplemented with 10 ml of acetonitrile
and sonicated for 5 min.
After removing the acetonitrile by blowing with nitrogen gas, peptides in the
digestion mixture were trapped on C18 resin packed in a ZipTipC18 (Millipore
Corporation, MA, USA), and eluted in 5 ml of 75% (v/v) methanol, 1% (v/v) formic
acid. Nano-ESI-MS for peptide-mass ®ngerprinting was performed using a Micromass Q-Tof system, equipped with a NanoFlow Probe Tip Type F (Micromass UK
Ltd., Manchester, UK). The peptide solution was put into a bolosilicate capillary tip
and subjected to electrospray ionization (ESI) at a ¯ow rate of 10 nl/min. The MS
spectrum was analyzed with the ProteinLynx software. Protein identi®cation through
a database search was carried out using the MS-FIT proteomic tool at the UCSF
web server, through the internet.

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Fig. 3. Identi®cation of spot protein by MS. The spot protein was transferred to a PVDF membrane, and digested
with lysylendoproteinase Lys-C. The mass spectrum of peptide fragments was obtained using a Micromass Q-Tof
MS system. The mass ®ngerprint database search was executed on the internet MS-FIT site. The ssp7001 was
identi®ed as phosphoprotein stathmin.

3. Results
3.1. Screening of age-related protein alteration by proteome pro®ling and differential
protein display
Alterations in the relative intensity of protein spots appearing on proteome pro®les, may
be the results of various molecular alterations that occur during aging. Quantitation of the
integrated optical density of each protein spot is required for the 2D gel electrophoresis of
protein, to screen out the molecular events that may be responsible for the functional
deterioration in senescent cells.
Coomassie Blue staining is the most reliable method for the quantitative demonstration

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of proteins on a gel slab. However, the sensitivity of Coomassie staining is not high
enough to detect minor components of cellular proteins. Autoradiography of [ 35S]methionine-labeled proteins shows the highest sensitivity. However, it is not applicable to the
detection of post-translational modi®cation of proteins. This is due to the fact that, only the
fresh proteins that are de novo synthesized during the period of labeling incubation are
detected by autoradiography. Cypro ruby ¯uorescent staining shows almost the same
sensitivity as silver staining, and quantitativity as Coomassie staining. Therefore, ¯uorescent staining will be included in the standard protocol of proteomics in the future. Silver
staining is a practical method for spot protein visualization in proteome analysis at present,
because it shows the highest sensitivity and practical reliability in relative quantitation for
differential protein display, as shown in Fig. 2.
In this 2D gel area, the transitional increase of spot protein ssp7001 was observed
around 65 PDL; at that level, the normal human diploid ®broblasts (TIG-3) transfer into
phase 3 of the replicative cell aging and the doubling time was delayed.
3.2. Identi®cation of spot protein by mass spectrometry in proteomics
In general, western blotting has been performed for the identi®cation of proteins in spots
for the last several years. However, the immunochemical technique was not applicable to
the unknown protein for which a speci®c antibody was not available. Co-electrophoresis is
another way to assign a spot to a candidate protein when its authentic protein standard
protein is available. In modern proteomics, a sequence database search queried with
peptide mass ®ngerprint data and/or partial sequence tag data obtained by MS, is generally
performed to identify proteins on 2D gel patterns. Fig. 3 shows an example of identi®cation of ssp7001 spot protein by peptide mass ®ngerprinting. The mass spectrum of Lys-C
digests of ssp7001 was recorded using a Micromass ESI-Q-Tof-MS system (MICROMASS UK, Manchester, UK). The database search was queried to MS-FIT in the Protein
Prospector Server at California University, and subsequently it was assigned to a microtubule associating phosphoprotein stathmin. When no candidate protein is hit by the
database search, because it is really novel, molecular cloning should be done to clarify
the meaning of the protein alteration in the aging process. In that case, Edman-degradation
microsequencing has an advantage over mass spectrometry in sequencing longer peptide
fragments of the protein in order to design proves and/or primers in molecular cloning of
its corresponding cDNA.
3.3. Determination of protein modi®cation by mass spectrometry in proteomics
Age-related protein alterations in heat stability and speci®c activity have been reported
by many groups (Holliday and Tarrant, 1972; Gershon and Gershon, 1973; Chetsanga and
Liskiwskyi, 1977; Pigeolet and Remacle 1991). Further, the accumulation of detergentinsoluble protein was also observed in aged cells and tissues (Yang and Wang, 1994). It
was suggested that these age-related alterations re¯ect post-translational modi®cations
such as oxidation by attacking of reactive oxygen species (Gordillo et al., 1989). Oxidative
modi®cation may produce carbonyl groups, ortho-tyrosine structures and methionine
sulfoxide residues that increase the hydrophobicity of the protein surface (Chao et al.,
1997). In the proteome analysis, structures of oxidative modi®cation on spot proteins,

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Fig. 4. Determination of methionine oxidation in human amyloid beta peptide (1±40) by MALDI-TOF-MS. The
sample solution was prepared as described in Methods. One-ml aliquot was applied to the MALDI target plate.
The mass spectrum was recorded with a Voyager-DE STR MALDI-TOF-MS system (PE Biosystems, Foster
City, CA) in a re¯ection mode. The spectrum indicated that the authentic preparation of human amyloid beta
peptide (1±40) contained both native and Met35-oxidized forms. Detected ions were all monovalent and the
15.96-Da mass shift was derived from the methionine oxidation.

isolated by 2D gel electrophoresis, can be determined by MS. Human amyloid beta
(Ab)1±40 peptide contains a methionine, and its oxidation induces the alteration of the
3D structure. The Met35-oxidized Ab shows a higher molecular mass of 16 Da than native
Ab, as shown in Fig. 4. Carbonylation does not result in a suf®cient shift in molecular mass
(21 Da); however, theoretically its dinitrophenylhydrazine derivatives gives an increase
of 180 Da.
4. Discussion
We have obtained an excellent methodology, proteomics, which is most suitable for
investigating protein factors in molecular mechanisms of cellular aging. Alterations in
gene expression that are the results of DNA damages, and accumulation of altered proteins
that are made by oxidative modi®cation can be detected by methods in proteomics. The
advanced method of two-dimensional gel electrophoresis, in which isoelectric focusing is

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carried out on an immobilized pH gradient for the ®rst dimensional separation, offers the
highest resolution of proteins. More than thousands of spots are detected on a gel slab in an
optimized condition. The proteome database of normal human diploid ®broblasts TIG-3
has been established, and is displayed on our Gerontology Informatics database: the PDLdependent alterations of protein spots are demonstrated here. Although the resolution of
2D gel electrophoresis is still not high enough to separate all the proteins in a cell extract, it
will be improved by partitioning the pH and molecular mass range using narrow pH range
IPG-IFE gel strips and various concentrations of acrylamide gel slabs for SDS-PAGE. The
multiplication of 2D gel electrophoresis for partitioning the range of separation may yield
better results than the simple enlargement of a gel size to cover the overall range of
separation. Identi®cation of the protein by making inquiries on the proteomic database
with a peptide mass ®ngerprint data, will be more successful after completion of the
genome-sequencing projects. Most post-translational modi®cations that are accumulated
in aged cells under attacks of reactive oxygen species (ROS), are easily detected by
proteomics. The extensive application of proteomics in the investigation of altered
proteins in aged cells has led to the next stage of research: the molecular mechanisms
of aging.
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