Identification of smoked Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) using PCR-restriction fragment length polymorphism of the p53 gene

Raw and smoked samples of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the p53 gene. DNA from S. salar and 0. mykiss was amplified by using primers flanking exons 5 to 6 of the p53 nuclear gene. PCR products of different length were obtained for each species (532 and 518 base pairs, respectively). Sequences of PCR products obtained from S. salar and O. mykiss were compared in the search for polymorphic restriction sites. The restriction fragments obtained with Eco RV, Hinf I, and Taq I endonucleases showed interspecific polymorphism, making it a useful method for identification of Atlantic salmon and rainbow trout.     

Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization--mass spectrometry, nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry, and tandem mass spectrometry

Optimal application of biological mass spectrometry (MS) in combination with two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of human cerebrospinal fluid (CSF) can lead to the identification of new potential biological markers of neurological disorders. To this end, we analyzed a number of 2-D PAGE protein spots in a human CSF pool using spot co-localization, N-terminal sequencing, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry (nanoLC-ESI-TOF-MS) with tandem MS switching. Our constructed CSF master contained 469 spots after image analysis and processing of 2-D gels. Upon visual inspection of our CSF master with the CSF pattern available on the ExPASy server, it was possible to locate and annotate 15 proteins. N-terminal sequence analysis and MALDI-MS peptide mass fingerprint analysis of both silver- and Coomassie Brilliant Blue (CBB) G-250-stained protein spots after in situ trypsin digest not only confirmed nine of the visually annotated spots but additionally resolved the identity of another 13 spots. Six of these proteins were not annotated on the 2-D ExPASy map: complement C3 alpha-chain (1321-1663), complement factor B, cystatin C, calgranulin A, hemoglobin beta-chain, and beta-2-microglobulin. It was clear that MALDI-MS identification from CBB G-250-stained, rather than from silver-stained, spots was more successful. In cases where no N-terminal sequence and/or no clear MALDI-MS result was available, nanoLC-ESI-TOF-MS and tandem MS automated switching was used to clarify and/or identify these protein spots by generating amino acid sequence tags. In addition, enrichment of the concentration of low-abundant proteins on 2-D PAGE was obtained by removal of albumin and immunoglobulins from the CSF pool using affinity chromatography. Subsequent analysis by 2-D PAGE of the fractionated CSF pool showed various new silver-stainable protein spots, of which four were identified by nanoLC-ESI-TOF-MS and tandem MS switching. No significant homology was found in either protein or DNA databases, indicating than these spots were unknown proteins.     

Cyano2Dbase updated: linkage of 234 protein spots to corresponding genes through N-terminal microsequencing.

The cyanobacterium Synechocystis sp. strain PCC6803 is an interesting model organism for preoteome study because it is a photosynthetic procaryote and its genomic sequence has already been determined at our institute. We thus initiated characterization of this organism from a proteomic viewpoint by exploiting two-dimensional (2-D) gel electrophoresis coupled with N-terminal protein sequencing. In a previous study, we linked 130 protein spots on two dimensional gels with the genes that encoded them. As an extension of the previous study, the number of protein spots linked to their corresponding genes was increased to 227 in this study by separately analyzing cyanobacterial proteins in four different fractions (soluble, insoluble, thylakoid membrane, and secretory protein fractions). The resultant updated 2-D protein-gene linkage database, named Cyano2Dbase, will serve as an indispensable tool in future cyanobacterial proteomic studies. From the data compiled in the Cyano2Dbase, we can extract many items of information concerning translation, posttranslational processing including characteristics of cyanobacterial signal sequences and modification of cyanobacterial proteins. The Cyano2Dbase is available to the public through the World Wide Web (http://www.kazusa.or.jp/tech/sazuka/cyano/pr oteome.html).     

Characterization and de novo sequencing of Atlantic salmon vitellogenin protein by electrospray tandem and matrix-assisted laser desorption/ionization mass spectrometry

Vitellogenin (VTG) is a protein produced by the liver of oviparous animals in response to circulating estrogens. In the plasma of males and immature females, VTG is undetectable. VTG has been used as a biomarker for exposure to endocrine disruptors in many species. In the present study, characterization of intact Atlantic salmon VTG was effected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). Tryptic digest peptides were analyzed by MALDI ToF MS to obtain a peptide mass fingerprint. De novo sequencing of the tryptic peptides used low-energy collisionally-induced dissociation (CID) in an electrospray ionization quadrupole-ToF orthogonal hybrid mass spectrometer (ESI Q-ToF MS/MS). The interpretation of the product-ion spectra obtained from the ESI Q-ToF MS/MS was done by Lutefisk, a computer-based software algorithm. The molecular mass of the intact protein was found to be 187335 Da. A total of 14 tryptic peptides were sequenced and compared with the complete rainbow trout VTG and the partial Atlantic salmon VTG sequences found in the Swiss-Prot database. De novo sequencing by CID MS/MS of 11 Atlantic salmon tryptic digest peptides with selected precursor ions at m/z 788.24, 700.20, 794.75, 834.31, 889.28, 819.79, 865.27, 843.81, 572.20, 573.66 and 561.68 showed high homology with the known sequence of rainbow trout VTG. The last two precursor peptide ions, found at m/z 573.66 and m/z 561.68, also specifically matched the known portion of the Atlantic salmon VTG sequence. Finally, three tryptic precursor peptide ions found at m/z 795.18, 893.28 and 791.05, provided product-ion spectra, which were exclusive to the unsequenced portion of the Atlantic salmon VTG.     

Proteome analysis of Corynebacterium glutamicum.

By the use of different Corynebacterium glutamicum strains more than 1.4 million tons of amino acids, mainly L-glutamate and L-lysine, are produced per year. A project was started recently to elucidate the complete DNA sequence of this bacterium. In this communication we describe an approach to analyze the C. glutamicum proteome, based on this genetic information, by a combination of two-dimensional (2-D) gel electrophoresis and protein identification via microsequencing or mass spectrometry. We used these techniques to resolve proteins of C. glutamicum with the aim to establish 2-D protein maps as a tool for basic microbiology and for strain improvement. In order to analyze the C. glutamicum proteome, methods were established to fractionate the C. glutamicum proteins according to functional entities, i.e., cytoplasm, membranes, and cell wall. Protein spots of the cytoplasmic and membrane fraction were identified by N-terminal sequencing, immunodetection, matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray ionization-mass spectrometry (ESI-MS). Additionally, a protocol to analyze proteins secreted by C. glutamicum was established. Approximately 40 protein spots were observed on silver-stained 2-D gels, 12 of which were identified.     

Proteomic analysis of the endoplasmic reticulum from developing and germinating seed of castor (Ricinus communis)

Endoplasmic reticulum (ER) has been prepared and analysed from germinating and developing castor bean endosperm. A combination of one- and two-dimensional (1-D and 2-D) gel electrophoresis was used to study the complexity of sample and protein differences between the two stages. The ER of the developing oilseed is central to the synthesis, sorting and storage of protein and lipid reserves while the germinating seed is concerned with their degradation. Sample complexity has been reduced by separation of ER proteins into lumenal, peripheral membrane and integral membrane subfractions. Membrane proteins pose specific problems in aggregation and binding to passive surfaces. We have overcome this by collection of membranes at density gradient interfaces and by silanization of plastic ware. Several major components have been identified from 1-D gels by N-terminal sequencing and matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprints. These include protein disulphide isomerase (PDI), calreticulin and developing-ER-specific oleate-12-hydroxylase involved in the biosynthesis of ricinoleic acid. In excess of 300 spots are detectable in each developmental fraction by high sensitivity 2-D gels. This is the first 2-D electrophoretic analysis of plant ER. These gels reveal significant differences between germinating and developing ER. Preparative loading 2-D gels of germinating ER have been run and 14 selected spots characterized by quadrupole time of flight tandem mass spectrometry (Q-TOF MS/MS). Ten of these proteins were assigned function on the basis of identity with existing castor database entries, or by homology with other species. Two proteins, aspartate proteinase precursor and N-carbamyl-L-aminohydrolase-like protein, appear to be absent from developing profiles. Most of the proteins identified are concerned with roles in protein processing and storage, and lipid metabolism which occur in the ER. Data from three of the assigned spots included unidentified peptides indicating the presence of more than one protein in these spots following 2-D electrophoresis. More extensive analysis will have to await developments in genomics but the basic separation technologies to simplify sample identity for a plant ER preparation have been established.     

Developing column material for the separation of serum amyloid P and C reactive protein from biological sources

In this study, we have investigated the isolation of serum amyloid P (SAP) and C‐reactive protein (CRP) from rainbow trout. It has recently been found that SAP is deposited in atherosclerotic lesions or neurofibrillary tangles, which are related to aging process and Alzheimer's disease. Given the importance of CRP, the CRP level in blood is becoming recognized as a potential means of monitoring cardiovascular risk. These two proteins, members of the pentraxin family of oligomeric serum proteins, were isolated from rainbow trout using N‐methacryloyl‐phosphoserine (MA‐pSer) immobilized poly (2‐hydroxy ethylmethacrylate) (PHEMA) cryogels as a column material in a fast protein liquid chromatography system. The separation process was verified in two steps. First, SAP and CRP proteins were isolated together from serum sample of rainbow trout using MA‐pSer/PHEMA cryogel columns. Second, SAP protein was separated chromatographically from CRP protein using the Ca²⁺ ion immobilized PHEMA cryogel column. According to the data, a new and effective technique has been developed for the isolation of SAP and CRP proteins from a biological source, rainbow trout. Finally, purified SAP and CRP were loaded using sodium dodecyl sulfate–polyacrylamide gel and western blot analysis to investigate the purity of chromatographically isolated SAP and CRP compared with commertial SAP and CRP. Copyright © 2014 John Wiley & Sons, Ltd.     

HPLC Quantification of astaxanthin and canthaxanthin in Salmonidae eggs

Astaxanthin and canthaxanthin are naturally occurring antioxidants referred to as xanthophylls. They are used as food additives in fish farms to improve the organoleptic qualities of salmonid products and to prevent reproductive diseases. This study reports the development and single‐laboratory validation of a rapid method for quantification of astaxanthin and canthaxanthin in eggs of rainbow trout (Oncorhynchus mykiss ) and brook trout (Salvelinus fontinalis М.). An advantage of the proposed method is the perfect combination of selective extraction of the xanthophylls and analysis of the extract by high‐performance liquid chromatography and photodiode array detection. The method validation was carried out in terms of linearity, accuracy, precision, recovery and limits of detection and quantification. The method was applied for simultaneous quantification of the two xanthophylls in eggs of rainbow trout and brook trout after their selective extraction. The results show that astaxanthin accumulations in salmonid fish eggs are larger than those of canthaxanthin. As the levels of these two xanthophylls affect fish fertility, this method can be used to improve the nutritional quality and to minimize the occurrence of the M74 syndrome in fish populations.     

Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry

Two-dimensional gel electrophoresis, bioinformatics, and mass spectrometry are key analysis tools in proteome analysis. The further characterization of post-translational modifications in gel-separated proteins relies fully on data obtained by mass spectrometric analysis. In this study, stress-induced changes in protein expression in Saccharomyces serevisiae were investigated. A total of eleven spots on a silver-stained two-dimensional (2-D) gel were identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping to represent C and/or N-terminal processed forms of enolase 2. The processing sites were determined by MALDI peptide mass mapping using a variety of proteolytic enzymes, by optimizing the sample preparation procedure and by specific labeling of all C-termini derived from in-gel digestion using a buffer containing 16O:18O (1:1). Out of eleven processed forms of enolase 2, six were fully characterized and the approximate processing sites identified for the remaining five.     

Proteome analysis of the culture environment supporting undifferentiated mouse embryonic stem and germ cell growth

The therapeutical interest of pluripotent cells and ethical issues related to the establishment of human embryonic stem cell (ESC) or embryonic germ cell (EGC) lines raise the understanding of the mechanism underlying pluripotency to a fundamental issue. Establishing a protein pluripotency signature for these cells can be complicated by the presence of unrelated proteins produced by the culture environment. Here, we have analyzed the environment supporting ESC and EGC growth, and established 2-D reference maps for each constituent present in this culture environment: mouse embryonic fibroblast feeder cells, culture medium (CM) and gelatin. The establishment of these reference maps is essential prior to the study of ESC and EGC specific proteomes. Indeed, these maps can be subtracted from ESC or EGC maps to allow focusing on spots specific for ESCs or EGCs. Our study led to the identification of 110 unique proteins from fibroblast feeder cells and 23 unique proteins from the CM, which represent major contaminants of ESC and EGC proteomes. For gelatin, no collagen-specific proteins were identified, most likely due to difficulties in resolution and low quantities. Furthermore, no differences were observed between naive and conditioned CM. Finally, we compared these reference maps to ESC 2-D gels and isolated 17 ESC specific spots. Among these spots, proteins that had already been identified in previous human and mouse ESC proteomes were identified but no apparent ESC-specific pluripotency marker could be identified. This work represents an essential step in furthering the knowledge of environmental factors supporting ESC and EGC growth.     

Development of an integrated approach for evaluation of 2-D gel image analysis: impact of multiple proteins in single spots on comparative proteomics in conventional 2-D gel/MALDI workflow

With 2-D gel mapping, it is often observed that essentially identical proteins migrate to different positions in the gel, while some seemingly well-resolved protein spots consist of multiple proteins. These observations can undermine the validity of gel-based comparative proteomic studies. Through a comparison of protein identifications using direct MALDI-TOF/TOF and LC-ESI-MS/MS analyses of 2-D gel separated proteins from cauliflower florets, we have developed an integrated approach to improve the accuracy and reliability of comparative 2-D electrophoresis. From 46 spots of interest, we identified 51 proteins by MALDI-TOF/TOF analysis and 108 proteins by LC-ESI-MS/MS. The results indicate that 75% of the analyzed spots contained multiple proteins. A comparison of hit rank for protein identifications showed that 37 out of 43 spots identified by MALDI matched the top-ranked hit from the ESI-MS/MS. By using the exponentially modified protein abundance index (emPAI) to determine the abundance of the individual component proteins for the spots containing multiple proteins, we found that the top-hit proteins from 40 out of 43 spots identified by MALDI matched the most abundant proteins determined by LC-MS/MS. Furthermore, our 2-D-GeLC-MS/MS results show that the top-hit proteins in 44 identified spots contributed on average 81% of the spots' staining intensity. This is the first quantitative measurement of the average rate of false assignment for direct MALDI analysis of 2-D gel spots using a new integrated workflow (2-D gel imaging, "2-D GeLC-MS/MS", and emPAI analysis). Here, the new approach is proposed as an alternative to traditional gel-based quantitative proteomics studies.     

Characterisation of the intact rainbow trout vitellogenin protein and analysis of its derived tryptic and cyanogen bromide peptides by matrix-assisted laser desorption/ionisation time-of-flight-mass spectrometry and electrospray ionisation quadrupole/time-of-flight mass spectrometry

Vitellogenin (VTG) is a protein produced by the liver of oviparous animals. It is being used as a biomarker for exposure to endocrine disruptors in many species. Rainbow trout Vtg has recently been sequenced by the conventional cDNA nucleotide approach. We focused on protein characterization of the intact protein and its derived tryptic and cyanogen bromide peptides by matrix-assisted laser desorption/ionisation and electrospray ionisation mass spectrometry. The molecular mass of the intact protein was found to be 183127 Da. A large number of unidentified peptide ions encourage further structural analysis to propose possible sequence variants and post-translational modifications.     

Profiling of Caenorhabditis elegans proteins using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry

The nematode Caenorhabditis elegans (C. elegans) is the first animal whose whole 97 Mb genome sequence, encoding ca. 19000 open reading frames (ORF's), has been essentially determined. We tried to establish a 2-DE map of the nematode proteome by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A soluble protein fraction of mixed stages of the worm, wild-type strain N2, was applied to 2-D PAGE. After Coomassie Brilliant Blue (CBB) staining, 1200 spots were detected and 140 major spots were excised from the gel and subjected to in-gel digestion with Achromobacter protease I (lysyl endopeptidase). Resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting for protein identification. With this approach we have obtained a two-dimensional electrophoresis (2-DE) protein map in which 69 spots were localized as landmarks for comparison of expression profiles to elucidate the basis of various biological events.     

Towards a two-dimensional proteome map of Mycoplasma pneumoniae

A Proteome map of the bacterium Mycoplasma pneumoniae was constructed using two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry (MS). M. pneumoniae is a human pathogen with a known genome sequence of 816 kbp coding for only 688 open reading frames, and is therefore an ideal model system to explore the scope and limits of the current technology. The soluble protein content of this bacterium grown under standard laboratory conditions was separated by 1-D or 2-D gel electrophoresis applying various pH gradients, different acrylamide concentrations and buffer systems. Proteins were identified using liquid chromatography-electrospray ionization ion trap and matrix-assisted laser desorption/ionization-MS. Mass spectrometric protein identification was supported and controlled using N-terminal sequencing and immunological methods. So far, proteins from about 350 spots were characterized with MS by determining the molecular weights and partial sequences of their tryptic peptides. Comparing these experimental data with the DNA sequence-derived predictions it was possible to assign these 350 proteins to 224 genes. The importance of proteomics for genome analysis was shown by the identification of four proteins, not annotated in the original publication. Although the proteome map is still incomplete, it is already a useful reference for comparative analyses of M. pneumoniae cells grown under modified conditions.     

Two-dimensional gel protein database of Saccharomyces cerevisiae (update 1999).

By proving the opportunity to visualize several hundred proteins at a time, two-dimensional (2-D) gel electrophoresis is an important tool for proteome research. In order to take advantage of the full potential of this technique for yeast studies, we have undertaken a systematic identification of yeast proteins resolved by this technique. We report here the identification of 92 novel protein spots on the yeast 2-D protein map. These identifications extend the number of protein spots identified on our yeast reference map to 401. These spots correspond to the products of 279 different genes. They have been essentially identified by three methods: gene overexpression, amino acid composition and mass spectrometry. These data can be accessed on the Yeast Protein Map server (htpp://www.ibgc.u-bordeaux2.fr/YPM).     

Long-term UVB Irradiation Affects the Immune Functions of Carp (Cyprinus carpio) and Rainbow Trout (Oncorhynchus mykiss)

The effects of long-term, low-dose ultraviolet B (UVB) radiation on immune functions of two fish species representing different taxonomic groups, carp ( Cyprinus carpio ) and rainbow trout ( Oncorhynchus mykiss ), were assessed in this study. The fish were exposed to 7, 20 or 60 mJ cm⁻² UVB three times per week, for 6 weeks. In carp, UVB exposure affected the respiratory burst activity of blood and head kidney phagocytes, differential blood leukocyte counts and blood chemistry. Phytohemagglutinin (PHA)-stimulated in vitro proliferation responses of blood and head kidney lymphocytes, however, remained unchanged. Rainbow trout tolerated the irradiations with fewer alterations, but significant changes were detected in blood chemistry and hematocrits of the irradiated fish. These results indicate that long-term exposure to low doses of UVB induces immunomodulation in fish, and that there are species-specific differences in sensitivity to irradiation.     

Rapid method for the measurement of circulating thyroid hormones in low volumes of teleost fish plasma by LC-ESI/MS/MS

Thyroid hormones are critical regulators of normal development and physiological functioning in all vertebrates. Radioimmunoassay (RIA) approaches have been the method of choice for measuring circulating levels of thyroid hormones in vertebrates. While sensitive, RIA-based approaches only allow for a single analyte measurement per assay, can lack concordance across platforms and laboratories, and can be prone to analytical interferences especially when used with fish plasma. Ongoing advances in liquid chromatography tandem mass spectrometry (LC/MS/MS) have led to substantial decreases in detection limits for thyroid hormones and other biomolecules in complex matrices, including human plasma. Despite these advances, current analytical approaches do not allow for the measurement of native thyroid hormone in teleost fish plasma by mass spectrometry and continue to rely on immunoassay. In this study, we developed a new method that allows for the rapid extraction and simultaneous measurement of total T4 (TT4) and total T3 (TT3) in low volumes (50 μL) of fish plasma by LC/MS/MS. Methods were optimized initially in plasma from rainbow trout (Oncorhynchus mykiss) and applied to plasma from other teleost fishes, including fathead minnows (Pimephales promelas), mummichogs (Fundulus heteroclitus), sockeye salmon (Oncorhynchus nerka), and coho salmon (Oncorhynchus kisutch). Validation of method performance with T4- and T3-spiked rainbow trout plasma at 2 and 4 ng/mL produced mean recoveries ranging from 82 to 95 % and 97 to 105 %, respectively. Recovery of ¹³C₁₂-T4 internal standard in plasma extractions was: 99?±?1.8 % in rainbow trout, 85?±?11 % in fathead minnow, 73?±?5.0 % in mummichog, 73?±?1.7 % in sockeye salmon, and 80?±?8.4 % in coho salmon. While absolute levels of thyroid hormones measured in identical plasma samples by LC/MS/MS and RIA varied depending on the assay used, T4/T3 ratios were generally consistent across both techniques. Less variability was measured among samples subjected to LC/MS/MS suggesting a more precise estimate of thyroid hormone homeostasis in the species targeted. Overall, a sensitive and reproducible method was established that takes advantage of LC/MS/MS techniques to rapidly measure TT4 and TT3 with negligible interferences in low volumes of plasma across a variety of teleost fishes.     

Protein synthesis in murine organs during postimplantation development detected by two-dimensional gel electrophoresis.

Mouse embryos were isolated from the uterus on days 10 to 11 of gestation and incubated in Dulbecco's modified Eagle's medium (DMEM) with [35S]methionine for 4 h. Subsequently, their hearts and the brains were dissected. The brain was divided into three parts, containing the telencephalon, mesencephalon, and myelencephalon. These tissues were then processed for two-dimensional (2-D) gel electrophoresis. Protein synthesis of the isolated tissues was analyzed for organ-and cell lineage-specific patterns. We studied proteins with isoelectric points (pI) ranging from 4 to 10 and relative molecular weights (M(r)) varying from 10000 to 200000 and found several significant quantitative and qualitative differences between the tissues and the developmental stages analyzed. In particular, we were able to distinguish between protein spots that we now attribute putatively to the corresponding embryonic organs. These differences may reflect some of the organ- and cell lineage-specific changes in protein synthesis and gene expression during early mammalian differentiation.     

Study of aluminium-exposed fish by scanning proton microprobe analysis

A scanning proton microprobe was used to study the aluminium distribution in gills of bullhead, pumpkinseed and rainbow trout exposed to aluminium at low pH. In the examined pumpkinseed gills, aluminium was located on the secondary lamellae edges. Aluminium hot spots were observed inside the tissue. The presence of aluminium coincided with elevated phosphorus levels. Element maps recorded in bullhead and rainbow trout failed to demonstrate the presence of aluminium. The technique of scanning transmission ion microscopy was used for sample visualisation as well as monitoring of the possible sample damage induced by particle induced X-ray emission-measurements.     

Age-dependent variations of cell response to oxidative stress: proteomic approach to protein expression and phosphorylation

We investigated the protein profiles of variously aged rat astrocytes in response to oxidative stress. After H2O2-exposure of cells at 100 microM for 30 min, the relative intensity of ten protein spots changed on two-dimensional (2-D) gels compared with control gels after silver staining. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis after in-gel digestion revealed that six of these spots corresponded to three kinds of proteins, each of which was composed of a protein and its modified form with a different isoelectric point (pI). These three proteins were identified as peroxiredoxins (PRDXs) II and III, and calpactin I light chain (p11). H2O2-exposure increased the intensity of the spot with lower pI and simultaneously decreased that of the spot with higher pI for both PRDXs II and III. In addition, the expression of annexin VII, S-adenosyl-L-homocysteine hydrolase, elongation factor II fragment (EF-II), and adenosine deaminase was increased by H2O2-exposure in astrocytes from variously aged rats. Using the Pro-Q Diamond staining, heat shock protein 60 kDa (Hsp 60) and alpha-tubulin were observed to be phosphorylated upon H2O2-exposure. While phosphorylation of alpha-tubulin was correlated positively with age, the changes in abundance of ten protein spots as described above were independent of age. These results suggest that aging does not suppress the responses aimed at limiting injury and promoting repair brought about by severe oxidative stress, and might affect cell dynamics including the formation of microtubules.