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Differential Proteome Analysis of Replicative Senescence in Rat Embryo Fibroblasts

2002; Elsevier BV; Volume: 1; Issue: 4 Linguagem: Inglês

10.1074/mcp.m100028-mcp200

1535-9484

Silvia Benvenuti, Rainer Cramer, Christopher C. Quinn, Jim Bruce, Marketa Zvelebil, S. Corless, Jacquelyn Bond, Alice Yang, Susan Hockfield, Alma L. Burlingame, Michael D. Waterfield, Parmjit Jat,

Microtubule and mitosis dynamics

Normal somatic cells undergo a finite number of divisions and then cease dividing whereas cancer cells are able to proliferate indefinitely. To identify the underlying mechanisms that limit the mitotic potential, a two-dimensional differential proteome analysis of replicative senescence in serially passaged rat embryo fibroblasts was undertaken. Triplicate independent two-dimensional gels containing over 1200 spots each were run, curated, and analyzed. This revealed 49 spots whose expression was altered more than 2-fold. Of these, 42 spots yielded positive protein identification by mass spectrometry comprising a variety of cytoskeletal, heat shock, and metabolic proteins, as well as proteins involved in trafficking, differentiation, and protein synthesis, turnover, and modification. These included gelsolin, a candidate tumor suppressor for breast cancer, and α-glucosidase II, a member of the family of glucosidases that includes klotho; a defect in klotho expression in mice results in a syndrome that resembles human aging. Changes in expression of TUC-1, -2, -4, and -4β, members of the TUC family critical for neuronal differentiation, were also identified. Some of the identified changes were also shown to occur in two other models of senescence, premature senescence of REF52 cells and replicative senescence of mouse embryo fibroblasts. The majority of these candidate proteins were unrecognized previously in replicative senescence. They are now implicated in a new role. Normal somatic cells undergo a finite number of divisions and then cease dividing whereas cancer cells are able to proliferate indefinitely. To identify the underlying mechanisms that limit the mitotic potential, a two-dimensional differential proteome analysis of replicative senescence in serially passaged rat embryo fibroblasts was undertaken. Triplicate independent two-dimensional gels containing over 1200 spots each were run, curated, and analyzed. This revealed 49 spots whose expression was altered more than 2-fold. Of these, 42 spots yielded positive protein identification by mass spectrometry comprising a variety of cytoskeletal, heat shock, and metabolic proteins, as well as proteins involved in trafficking, differentiation, and protein synthesis, turnover, and modification. These included gelsolin, a candidate tumor suppressor for breast cancer, and α-glucosidase II, a member of the family of glucosidases that includes klotho; a defect in klotho expression in mice results in a syndrome that resembles human aging. Changes in expression of TUC-1, -2, -4, and -4β, members of the TUC family critical for neuronal differentiation, were also identified. Some of the identified changes were also shown to occur in two other models of senescence, premature senescence of REF52 cells and replicative senescence of mouse embryo fibroblasts. The majority of these candidate proteins were unrecognized previously in replicative senescence. They are now implicated in a new role. Cancer arises as a consequence of the accumulation of multiple independent mutations in genes that regulate cell proliferation and survival (1.Hanahan D. Weinberg R.A. The hallmarks of cancer.Cell. 2000; 100: 57-70Google Scholar). The acquisition of an unlimited proliferative potential has been proposed to be one of the critical steps in this process, because normal cells can only undergo a finite number of divisions when cultured in vitro before undergoing replicative senescence (2.Hayflick L. The cell biology of aging.Clin. Geriatr. Med. 1985; 1: 15-27Google Scholar). Even though replicative senescence has been studied extensively and can be overcome by immortalizing genes, the underlying molecular basis is still not understood fully. In human somatic cells telomere shortening is a critical component of the machinery that counts the number of cell divisions and therefore entry into senescence. It was proposed initially that reconstitution of telomerase activity resulting in maintenance of telomeres was sufficient for immortalization of human somatic cells, but others have found that this is not sufficient and requires additional activities such as those that can be provided by the SV40 (simian virus 40) large T antigen or inactivation of the pRB/p16 INK4 pathway (3.Counter C.M. Hahn W.C. Wei W. Caddle S.D. Beijersbergen R.L. Lansdorp P.M. Sedivy J.M. Weinberg R.A. Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14723-14728Google Scholar, 4.Hahn W.C. Counter C.M. Lundberg A.S. Beijersbergen R.L. Brooks M.W. Weinberg R.A. Creation of human tumour cells with defined genetic elements.Nature. 1999; 400: 464-468Google Scholar, 5.Kiyono T. Foster S.A. Koop J.I. McDougall J.K. Galloway D.A. Klingelhutz A.J. Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells.Nature. 1998; 396: 84-88Google Scholar). Further studies have now shown that in freshly isolated human mammary fibroblasts and endothelial cells, reconstituted telomerase activity was sufficient neither for immortalization nor maintenance of the immortal state in cell lines that had been immortalized with a combination of the SV40 T antigen and the catalytic subunit of telomerase (6.O'Hare M.J. Bond J. Clarke C. Takeuchi Y. Atherton A.J. Berry C. Moody J. Silver A.R. Davies D.C. Alsop A.E. Neville A.M. Jat P.S. Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 646-651Google Scholar). Inactivation of SV40 T antigen in these cells resulted in a rapid and irreversible cessation of cell growth and entry into senescence. Even though telomere shortening cannot be demonstrated in rodent cells, they, too, have a finite life span. In contrast to human cells this can be overcome readily in rodent cells by either the exogenous introduction of any member of the family of viral and cellular immortalizing genes, such as SV40 T antigen, or even by spontaneous mutation. Interestingly like the human cells immortalized with SV40 T antigen and hTERT, rodent cells expressing SV40 T antigen proliferate indefinitely and are absolutely dependent upon its continued expression for maintenance of growth (7.Jat P.S. Sharp P.A. Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature.Mol. Cell. Biol. 1989; 9: 1672-1681Google Scholar). Inactivation also results in a rapid and irreversible cessation of growth and entry into senescence (8.Gonos E.S. Burns J.S. Mazars G.R. Kobrna A. Riley T.E. Barnett S.C. Zafarana G. Ludwig R.L. Ikram Z. Powell A.J. Jat P.S. Rat embryo fibroblasts immortalized with simian virus 40 large T antigen undergo senescence upon its inactivation.Mol. Cell. Biol. 1996; 16: 5127-5138Google Scholar). Moreover, we have shown that primary mouse embryo fibroblasts are able to measure their proliferative life span even in the presence of SV40 T antigen at the normal rate (9.Ikram Z. Norton T. Jat P.S. The biological clock that measures the mitotic life-span of mouse embryo fibroblasts continues to function in the presence of simian virus 40 large tumor antigen.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6448-6452Google Scholar). Taken together these results have raised the possibility that the non-telomere shortening-dependent regulatory components of the finite proliferative life span may be conserved between human and rodent cells and that human cells may have acquired telomere shortening as a further control mechanism. Replicative senescence is an asynchronous process whereby a growing culture gives rise to an irreversibly arrested culture. The model systems that are commonly used for its study involve the isolation and serial in vitro cultivation of primary fibroblasts. Initially these cells proliferate exponentially but cease dividing after some passages, at which point the cell numbers no longer increase. The loss of proliferative potential in such heterogeneous cultures of primary cells is asynchronous. When these cells have reached the end of their in vitro mitotic lifespan, they can be maintained and remain metabolically active but cannot be induced to undergo new rounds of cell division (10.Cristofalo V.J. Phillips P.D. Sorger T. Gerhard G. Alterations in the responsiveness of senescent cells to growth factors.J. Gerontol. 1989; 44: 55-62Google Scholar). In such model systems, the culture as a whole divides initially and undergoes growth arrest toward the end; however there can be growth-arrested cells in the early passages and dividing cells toward the later passages. The senescent phenotype is dominant as fusions of senescent cells with dividing cells give rise to senescent cells (11.Pereira-Smith O.M. Robetorye S. Ning Y. Orson F.M. Hybrids from fusion of normal human T lymphocytes with immortal human cells exhibit limited life span.J. Cell. Physiol. 1990; 144: 546-549Google Scholar). Even though a variety of traditional approaches have been utilized to try to identify the underlying changes that are the cause of senescence, this process is still not understood fully, probably because these procedures were insufficient to analyze comprehensively such a complex process. New approaches to address complex biological systems include DNA microarrays that monitor global changes in mRNA expression (12.Fodor S.P. Rava R.P. Huang X.C. Pease A.C. Holmes C.P. Adams C.L. Multiplexed biochemical assays with biological chips.Nature. 1993; 364: 555-556Google Scholar). However studies in Saccharomyces cerevisiae and human liver have suggested that mRNA levels may correlate poorly with corresponding protein levels (13.Anderson L. Seilhamer J. A comparison of selected mRNA and protein abundances in human liver.Electrophoresis. 1997; 18: 533-537Google Scholar, 14.Gygi S.P. Rochon Y. Franza B.R. Aebersold R. Correlation between protein and mRNA abundance in yeast.Mol. Cell. Biol. 1999; 19: 1720-1730Google Scholar), and mRNA-based assays are unable to detect changes in protein level because of stability and changes in post-translational modifications. Another approach would be to analyze changes in protein expression that have the potential for resolution of all expressed proteins in a cell (proteome), which can then be identified by mass spectrometry. Even though not all proteins may be resolved by two-dimensional (2-D) 1The abbreviations used are: 2-D, two-dimensional; REF, rat embryo fibroblast; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; HPLC, high pressure liquid chromatography; ESI-MS/MS, electrospray ionization tandem mass spectrometry; RT, reverse transcriptase; HSP, heat shock protein; 1-D, one-dimensional. gels and also may not be identified by mass spectrometry, we have initiated a differential proteomic approach to study replicative senescence. This was done, because it has the potential for identifying changes in protein expression, post-translational modification, stability, and even changes in cellular localization. Furthermore, we have chosen to study the protein expression profiles of serially passaged rat embryo fibroblasts (REFs), rather than primary human fibroblasts, to minimize differences because of epigenetic variation between cells obtained from different donors and also, because human fibroblasts have much longer finite proliferative lifespan in vitro. Human fibroblasts are capable of undergoing 50–60 divisions before undergoing replicative senescence, in contrast to 20–30 divisions for rodent embryo fibroblasts. The issue of epigenetic variation was critical, because we needed to be able to go back and repeat the passaging with freshly isolated identical cells to prepare protein extracts for validation of the proteome analysis and also extraction of RNA for determining whether changes at the protein level correlate with changes at the RNA level. Cells that are cultivated serially after freezing exhibit an altered finite mitotic life span. Changes in protein profiles upon cellular senescence were monitored by high resolution 2-D polyacrylamide gel electrophoresis, and differentially expressed protein spots were identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nano-HPLC electrospray ionization tandem mass spectrometry (ESI-MS/MS). This analysis identified 49 spots whose expression changed more than 2-fold upon replicative senescence; 32 of these spots were up-regulated, 12 were down-regulated, and five displayed an altered migration pattern. The majority of these proteins were unrecognized previously in replicative senescence. They are now implicated in a new role. REFs were prepared from 12–13-day-old Sprague-Dawley rat embryos, cultured, and passaged serially. All cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. REF52 cells obtained from Scott Lowe were propagated in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. All media and components were obtained from Invitrogen. 2-D gels were prepared and run as described previously (15.Page M.J. Amess B. Townsend R.R. Parekh R. Herath A. Brusten L. Zvelebil M.J. Stein R.C. Waterfield M.D. Davies S.C. O'Hare M.J. Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12589-12594Google Scholar). Staining, scanning, and curation of primary images with subsequent analysis to identify differential spots were all carried out by previously published procedures (15.Page M.J. Amess B. Townsend R.R. Parekh R. Herath A. Brusten L. Zvelebil M.J. Stein R.C. Waterfield M.D. Davies S.C. O'Hare M.J. Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12589-12594Google Scholar). Identifications of all differentially expressed proteins utilized a standard approach using MALDI-MS and if necessary ESI-MS/MS. Differential spots were excised from one of the triplicate gels, which showed the highest expression level for each spot. Prior to mass spectrometry, tryptic in-gel digests were carried out on all samples using a protocol similar to already published procedures (e.g. donatello.ucsf.edu/ingel.html; see Ref. 16.Wilm M. Shevchenko A. Houthaeve T. Breit S. Schweigerer L. Fotsis T. Mann M. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.Nature. 1996; 379: 466-469Google Scholar). Details of the procedures for peptide mass mapping by matrix-assisted laser desorption/ionization mass spectrometry and peptide sequencing by nano-HPLC electrospray ionization tandem mass spectrometry are provided as Supplementary Material. Cell lysates for immunoblotting were prepared in RIPA buffer (150 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris-HCl, pH 8.0). Protein concentrations were determined using the Bradford reagent (Bio-Rad). 30 μg of each protein lysate was fractionated on an SDS polyacrylamide gel, transferred to Hybond C membrane, and probed with the following antibodies: α-glucosidase (StressGene), HSP27 Ab-1 (NeoMarkers), HSP70 W27 (Santa Cruz Biotechnology), CDC47 (NeoMarkers), gelsolin (kindly provided by Helen Yin), cyclin A Ab-4 (Oncogene Science), and p19ARF (Abcam). First strand cDNA was prepared from 2 μg of total RNA using SuperScriptTM II Moloney murine leukemia virus RNaseH− reverse transcriptase from Invitrogen according to the manufacturer's instructions. RNA was denatured (65°C for 5 min) in the presence of an oligo(dT) primer (0.5 μg; Promega) and dNTPs (0.5 mm; Promega) and quickly chilled on ice. Ribonuclease inhibitor (RNasin; Promega) and dithiothreitol (100 mm) were then added, together with 1× first strand buffer, and incubated at 42°C for 2 min. Moloney murine leukemia virus RNaseH− reverse transcriptase (200 units) was added, and reactions were incubated at 42°C for 50 min, followed by heat inactivation at 70°C for 15 min. For each gene, an optimal cycle number was established that enabled the bands to be visible on a gel but did not result in saturation of the amplification procedure. This was done partially by confirming that the same relative intensities were obtained when the cycle number was increased by two cycles. The sequences of the primers used for PCRs, along with the product size, cycle numbers, and annealing temperatures, are provided as Supplementary Material. All PCR reactions were carried out in 50 μl and contained 1 μl of first strand cDNA. They also contained 0.5 μg of each oligonucleotide primer, 2.5 units of Thermus aquaticus (Taq) DNA polymerase (Promega), 1× PCR buffer (10 mm Tris-HCl, pH 9, 50 mm KCl, 0.1% (w/v) Triton X-100 (10× PCR buffer provided by Promega)), 0.5 mm dNTPs (Promega), and 1.5–2.5 mm MgCl2 (Promega). The optimum MgCl2 concentration was determined for each primer set and is also provided as Supplementary Material. A 5-min 95°C denaturation step was used prior to amplification. The amplification parameters were denaturation at 94°C for 1 min, annealing at the specific temperature for each primer pair for 1 min, and extension at 72°C for 1 min, with a final extension of 5 min at 72°C after the last cycle. For each PCR, appropriate controls were carried out to check for nonspecific amplification. All PCR primers were designed using Primer3 software (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). BOSC23 cells (1.75 × 106) were plated in a 6-cm dish and transfected 24 h later with 3 μg of either pBabePuroEJras or pBabePuro using FuGENE (Roche Molecular Biochemicals). 48 h after transfection, the virus-containing medium was removed, filtered (though a 0.45 μm filter), and used to infect REF52 cells in the presence of 8 μg/ml polybrene (Aldrich). REF52 were plated at 8 × 105 cells per 10-cm dish and incubated overnight prior to infection. For the infection, the culture medium was replaced by 2 ml of virus-containing medium for 2 h. The infection was repeated a second time 6 h later. Sixteen h later, the infected cultures were subjected to 2 μg/ml puromycin selection. The medium was changed on the third day, and the infected cells were extracted with RIPA buffer after 7 days. REFs were prepared from 12–13-day-old Sprague-Dawley rat embryos and passaged using the 3T3 passaging regime of Todaro and Green (17.Todaro G.J. Green H. Quantitative studies of the growth of mouse embryo cells in culture and their development into established cell lines.J. Cell Biol. 1963; 17: 299-313Google Scholar). Cells were passaged serially until they ceased to divide and cell numbers no longer increased, which takes about five passages. Cultures were continually examined microscopically to ensure that there were no mitotic cells visible when passaging was ceased. This involved plating 2.6 × 106 cells per 15-cm dish. Cells were plated on day 0 and allowed to adhere, the medium was changed on day 1, and the cells passaged on day 3. For preparation of cell lysates, cells were harvested on day 2. In this way all lysates were prepared from cultures that were in fresh medium and subconfluent and thus not quiescent. This was done to ensure that only changes in expression because of senescence were being examined. Triplicate independent total protein extracts prepared from three serially passaged independent dishes of REFs at P2 (proliferating cells), P3, P4, and P5 (senescent cells) were fractionated on 2-D gels to visualize changes in the proteome. The fractionated proteins were detected by staining the gels with the fluorescent dye OGT MP17, followed by scanning at a detection level of less than 1 ng of protein. A representative 2-D gel for P2 and P5 senescent REFs is shown in Fig. 1. The triplicate scanned gels were then subjected to triplicate curation in which the gel with the most spots (detected using MELANIE II software) was chosen as the reference gel, and the two other gels were called gel II and III, respectively. First gel II was curated against the reference gel and then gel III was curated against it. Next gel II was curated against gel III. Although triplicate curation is a time-consuming procedure, it allows identification of differential features using only the reference gels. However it is necessary as a follow-up to check manually that all features expressed differentially in the reference gel are also differential in the other two gels within the triplicate. Only protein spots that changed more than 2-fold in magnitude, in the same direction (i.e. up or down), and were observed in all three gels were considered. The pI and molecular weight of each spot was calculated by bilinear interpolation between landmark spots on each image that had been calibrated previously with respect to Escherichia coli proteins. After curation, each gel was found to comprise over 1200 spots. Moreover, the gels were very reproducible among each triplicate (the % of homology among the gels was always above 90%, shown in Table I).Table IPercent homology between the triplicate gelsReference gelGel IIGel IIIP210098.697.1P310098.099.0P410095.093.0P510096.096.1 Open table in a new tab The detected spots were then submitted to CHIMAP to identify the differentials. CHIMAP is a newly developed program that calculates a differential value between a large series of matched features either as a percentage change or a -fold change and represents them graphically (18.Harris R.A. Yang A. Stein R.C. Lucy K. Brusten L. Herath A. Parekh R. Waterfield M.D. O'Hare M.J. Neville M.A. Page M.J. Zvelebil M.J. Cluster analysis of an extensive human breast cancer cell line protein expression map database.Proteomics. 2002; 2: 212-223Google Scholar). It generally uses the Ward's minimum variance method although other agglomeration methods can be selected within the program. The comparison of growing versus senescent REFs identified 49 spots that were reproducibly regulated differentially, of which 32 were up-regulated, 12 were down-regulated, and five shifted in their position of migration (shown in Table II). Representative features that correspond to each type of differential are shown in Fig. 2. We also examined expression of the differential spots, in P3 and P4 gels, to determine whether the changes were also observed at these passages and whether they occurred stepwise or processively (shown in Supplemental Material, Table I).Table IIProtein annotationsUp-regulated featuresMALDI-MS spot identificationESI spot identificationIDMWpIF.C. P5 vs. P21021253865.48+3.2Alanyl-tRNA synthetase (h)1041253866.37+2.32-Oxoglutarate dehydrogenase precursor (h)1081242406.53>10*O-GlcNAc transferase p110 subunit (r)1101231055.53>10105-kDa heat shock protein (m)1221143905.57>10α-Glucosidase II, α subunit (m)1271133455.82>10α-Glucosidase II, α subunit (m)α-Glucosidase II, α subunit (m)158983055.01+2.31779333486.52+2.2Lysyl hydroxylase isoform 2 (m)185916084.96>10Heat shock protein 90-α (h)190916085.66+4.8Gelsolin (h and m) + myosin heavy chain (c)223837765.31>10248810627.08>10290748336.64+2.0Transferrin (b)369678815.90>10426647126.19+2.9Vesicle transport-related protein (RA410) (r)493607675.76+2.02-Oxoglutarate dioxygenase γ-butyrobetaine (r)495607676.47+3.3Chaperonin-containing TCP-1, γ-subunit (m) + TUC-4 (m)497604626.06+2.0Seryl-tRNA synthetase (h)Seryl-tRNA synthetase (h) + T complex protein 1, α (r)504601586.94+2.2MPAST1 (m)MPAST (h) + lamin A or C (r)677478885.57+3.0ERF1 (h) + probable ATP-dependent RNA helicase p47 (r)713451105.14+3.0Similar to cdc37 (r)773453866.54+3.1Elongation factor-1-γ (h)Elongation factor-1-γ (h)902401266.58+2.0Arp2 (h)Arp2 (h) + hnRNP-E2 (h)907400105.71+2.126 S proteasome subunit p40.5 (m)26 S proteasome subunit p40.5 (h)1075478885.79+2.0Isopentenyl diphosphate dimethylallyl diphosphate isomerase (r)1076339867.222.0CLP36 (r)1093233435.84>10Thiol-specific antioxidant (r)1095333856.42+2.01124323295.34+2.0*1264257896.05>10*1302241775.58+3.0Heat shock 27 protein (r)1319233705.57>10*Down-regulated featuresMALDI-MS spot identificationESI spot identificationIDMWpIF.C. P2 vs. P5151589866.38+2.0*153798744.76+2.5Sec23 protein (h) + ischemia-responsive 94-kDa protein (r)Sec23 protein (h) + ischemia-responsive 94-kDa protein (h) + HSP70 (h)278648585.09+3.2Preimmunoglobulin heavy chain binding protein (r)304633296.09+2.4TUC-2 (r)460532637.39+2.0IMP dehydrogenase (m)736392126.13+2.630-kDa protein (h)863343045.41+4.7Tubulin β chain 15 (r)882341725.57+3.9Transitional endoplasmic reticulum ATPase (r)1054253015.01+2.0ρ GDP dissociation inhibitor (b)ρ GDP dissociation inhibitor (h and b)1239141895.66+2.2Proteasome subunit RC10-II (r)Proteasome subunit RC10-II (r) + PRx III (r)1295244255.87+2.0PRx IV (r)1299245495.78+2.1Features that shift in their migrationMALDI-MS spot identificationESI spot identificationIDMWpI331711996.67Moesin (r)332711996.56Moesin (r) + guanosine 5′-monophosphate synthetase ? (h)1117220146.191348221346.20Proteasome subunit RC10-II (r)Proteasome subunit RC10-II (r)1351221346.10PRx III (r) Open table in a new tab Analysis of the differentially regulated spots by MALDI-MS yielded 24 single positive identifications, two double positive identifications, six single putative identifications, one double putative identification, and one double mixed (positive and putative) identification (shown in Table II, and presented in greater detail as Supplemental Material, Table II). Five spots yielded only keratin, and in four cases, more than 20 peptide ion signals were obtained, but database searches with these peptide mass lists did not identify any proteins. For the remaining six of the 49 differential spots, the mass spectra recorded exhibited not more than 10 peptides; these data were classified as insufficient for peptide mass mapping. From all identified proteins only 18 proteins were identified as rat proteins, nine were mouse, nine were human, and one was bovine. Interestingly, five proteins identified as non-rat proteins have rat homologues in the NCBI protein database suggesting either extensive nucleotide polymorphisms or incorrect database entries for the rat protein sequences. ESI-MS/MS analysis was performed in all cases where peptide mass mapping by MALDI-MS was unsuccessful or ambiguous. In addition, a few spots with sufficient MALDI-MS data for protein identification were analyzed by ESI-MS/MS to verify the identification by MALDI-MS peptide mass mapping. Almost two-thirds of the excised spots were analyzed by nano-HPLC ESI-MS/MS. The results for the protein identification by ESI-MS/MS (false positives excluded) are also summarized in Table II and presented in greater detail as Supplemental Material, Tables III and IV. From all proteins identified by searching the NCBI protein database only 15 proteins were identified as rat proteins, whereas three were from mouse, one was from chicken, one was from Chinese hamster, and 10 were human. MS/MS data from two spots identified human proteins with some peptides giving matches to the mouse and bovine homologues that did not match the human protein. Similarly, MS/MS data from one other spot resulted in peptide matches not matching the rat protein but rather the Chinese hamster homologue. The chicken myosin heavy chain identified in spot 190 was quite unusual, because a very close rat homologue exists in the NCBI protein database. However, the peptides for which the MS/MS data were obtained cover regions with little homology suggesting that the rat homologue remains to be identified. Positive protein identifications obtained by MALDI-MS peptide mass mapping were confirmed in all cases where ESI-MS/MS was employed. All putatively identified proteins by MALDI-MS peptide mass mapping were also confirmed by ESI-MS/MS whenever sufficient data were obtained. In addition to these confirmations three more proteins were identified, t-complex polypeptide in sample 497, lamin A or C2 in sample 504, and heat shock protein 27 in sample 1295. For five previously unidentified spots, positive protein identification was obtained. The comparison of the results from the MALDI-MS and ESI-MS/MS data demonstrated that both techniques show comparable sensitivity in protein identification. Furthermore, this comparison also showed that the criteria applied for protein identification were reliable and gave virtually no false identifications, with a minimal loss in analytical sensitivity. The differentially expressed proteins comprised a variety of cytoskeletal, heat shock, and metabolic proteins, as well as proteins involved in trafficking, differentiation, and protein synthesis, turnover, and modification. For the purposes of the discussion below, we have assumed that all proteins identified by mass spectrometry for each spot were expressed differentially. However this may clearly not be the case for spots that yielded multiple proteins; for these spots Western blot analysis using specific antibodies will be required to determine the identity of the differential protein. A number of the proteins identified, like gelsolin, β-tubulin, and Arp2 (actin-related protein 2), have been shown previously to be involved in cytoskeletal scaffolding. Most of these proteins were up-regulated during senescence; however β-tubulin was down-regulated. It is perhaps not surprising that proteins involved in the cytoskeleton show changes in expression upon replicative senescence, because senescent cells are very different morphologically from growing cells; they have the classic fried egg morphology of senescent cell