The physiological role of CPR1 in Saccharomyces cerevisiae KNU5377 against menadione stress by proteomics

In order to understand the functional role of CPR1 in Saccharomyces cerevisiae KNU5377 with regard to its multi-tolerance characteristics against high temperatures, inorganic acids, and oxidative stress conditions, whole cellular proteins were analyzed via liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This procedure was followed by two-dimensional (2-D) gel electrophoresis. Under menadione stress conditions, the 23 upregulated proteins were clearly identified only in the wildtype strain of KNU5377. Among the proteins, Sod1p, Tsa1p, Ahp1, Cpr1p, Cpr3, Ssb2p, and Hsp12p were identified as components of antioxidant systems or protein-folding related systems. The CPR1 protein could not be completely detected in the cpr1Delta mutant of KNU5377 and the other upregulated proteins in the wild-type strain evidenced a clear correlation with the results of immunoblot analysis. Moreover, a reduction in growth patterns (about 50%) could be observed in the cpr1Delta mutant, as compared with that of the wild-type strain under mild MD stress conditions. These results indicate that the upregulation of CPR1 may contribute to tolerance against MD as an inducer of oxidative stress.     

Heat shock causes oxidative stress and induces a variety of cell rescue proteins in Saccharomyces cerevisiae KNU5377

In this study, we attempted to characterize the physiological response to oxidative stress by heat shock in Saccharomyces cerevisiae KNU5377 (KNU5377) that ferments at a temperature of 40 degrees C. The KNU5377 strain evidenced a very similar growth rate at 40 degrees C as was recorded under normal conditions. Unlike the laboratory strains of S. cerevisiae, the cell viability of KNU5377 was affected slightly under 2 hours of heat stress conditions at 43 degrees C. KNU5377 evidenced a time-dependent increase in hydroperoxide levels, carbonyl contents, and malondialdehyde (MDA), which increased in the expression of a variety of cell rescue proteins containing Hsp104p, Ssap, Hsp30p, Sod1p, catalase, glutathione reductase, G6PDH, thioredoxin, thioredoxin peroxidase (Tsa1p), Adhp, Aldp, trehalose and glycogen at high temperature. Pma1/2p, Hsp90p and H+-ATPase expression levels were reduced as the result of exposure to heat shock. With regard to cellular fatty acid composition, levels of unsaturated fatty acids (USFAs) were increased significantly at high temperatures (43 degrees C), and this was particularly true of oleic acid (C18:1). The results of this study indicated that oxidative stress as the result of heat shock may induce a more profound stimulation of trehalose, antioxidant enzymes, and heat shock proteins, as well as an increase in the USFAs ratios. This might contribute to cellular protective functions for the maintenance of cellular homeostasis, and may also contribute to membrane fluidity.     

Decarbonylated cyclophilin A Cpr1 protein protects <Emphasis Type="BoldItalic">Saccharomyces cerevisiae</Emphasis> KNU5377Y when exposed to stress induced by menadione

Cyclophilins are conserved cis–trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. The accumulation of Cpr1 protein to menadione in Saccharomyces cerevisiae KNU5377Y suggests a possibility that this protein may participate in the mechanism of stress tolerance. Stress response of S. cerevisiae KNU5377Y cpr1Δ mutant strain was investigated in the presence of menadione (MD). The growth ability of the strain was confirmed in an oxidant-supplemented medium, and a relationship was established between diminishing levels of cell rescue enzymes and MD sensitivity. The results demonstrate the significant effect of CPR1 disruption in the cellular growth rate, cell viability and morphology, and redox state in the presence of MD and suggest the possible role of Cpr1p in acquiring sensitivity to MD and its physiological role in cellular stress tolerance. The in vivo importance of Cpr1p for antioxidant-mediated reactive oxygen species (ROS) neutralization and chaperone-mediated protein folding was confirmed by analyzing the expression changes of a variety of cell rescue proteins in a CPR1-disrupted strain. The cpr1Δ to the exogenous MD showed reduced expression level of antioxidant enzymes, molecular chaperones, and metabolic enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH)- or adenosine triphosphate (ATP)-generating systems. More importantly, it was shown that cpr1Δ mutant caused imbalance in the cellular redox homeostasis and increased ROS levels in the cytosol as well as mitochondria and elevated iron concentrations. As a result of excess ROS production, the cpr1Δ mutant provoked an increase in oxidative damage and a reduction in antioxidant activity and free radical scavenger ability. However, there was no difference in the stress responses between the wild-type and the cpr1Δ mutant strains derived from S. cerevisiae BY4741 as a control strain under the same stress. Unlike BY4741, KNU5377Y Cpr1 protein was decarbonylated during MD stress. Decarbonylation of Cpr1 protein in KNU5377Y strain seems to be caused by a rapid and efficient gene expression program via stress response factors Hsf1, Yap1, and Msn2. Hence, the decarbonylated Cpr1 protein may be critical in cellular redox homeostasis and may be a potential chaperone to menadione.     

Proteomic research reveals the stress response and detoxification of yeast to combined inhibitors

The tolerant mechanism of yeast to the combination of three inhibitors (furfural, phenol and acetic acid) was investigated using 2-DE combined with MALDI-TOF/TOF-MS. The stress response and detoxification related proteins (e.g., Ahp1p, Hsp26p) were expressed higher in the tolerant yeast than in the parental yeast. The expressions of most nitrogen metabolism related proteins (e.g. Gdh1p, Met1p) were higher in the parental yeast, indicating that the tolerant yeast decreases its nitrogen metabolism rate to reserve energy, and possesses high resistance to the stress of combined inhibitors. Furthermore, upon exposure to the inhibitors, the proteins related to protein folding, degradation and translation (e.g., Ssc1p, Ubp14p, Efb1p) were all significantly affected, and the oxidative stress related proteins (e.g., Ahp1p, Grx1p) were increased. Knockdown of genes related to the oxidative stress and unfolded protein response (Grx1, Gre2, Asc1) significantly decreased the tolerance of yeast to inhibitors, which further suggested that yeast responded to the inhibitors mainly by inducing unfolded protein response. This study reveals that increasing the detoxification and tolerating oxidative stress, and/or decreasing the nitrogen metabolism would be promising strategies in developing more tolerant strains to the multiple inhibitors in lignocellulose hydrolysates.     

A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification

A two-dimensional liquid-phase separation scheme coupled with mass spectrometry (MS) is presented for proteomic analysis of cell lysates from normal and malignant breast epithelial cell lines. Liquid-phase separations consist of isoelectric focusing as the first dimension and nonporous silica reverse-phase high-performance liquid chromatography (NPS-RP-HPLC) as the second dimension. Protein quantitation and mass measurement are performed using electrospray ionization-time of flight MS (ESI-TOF MS). Proteins are identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS) and MALDI-quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS). Two pH regions with 50-60 unique proteins in each pH range were chosen for analysis. Mass maps were created that allowed visualization of protein quantitation differences between normal and malignant breast epithelial cells. Of the approximately 110 unique proteins observed from mass mapping experiments over the limited pH range, 40 (36%) were positively identified by peptide mass fingerprinting and assigned to bands in the mass maps. Of these 40 proteins, 22 were more highly expressed in one or more of the malignant cell lines. These proteins represent potential breast cancer biomarkers that could aid in diagnosis, therapy, or drug development.     

Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p

Urm1p is a ubiquitin-related protein that serves as a posttranslational modification of other proteins. Urm1p conjugation has been implicated in the budding process and in nutrient sensing. Here, we have identified the first in vivo target for the urmylation pathway as the antioxidant protein Ahp1p. The attachment of Urm1p to Ahp1p requires the E1 for the urmylation pathway, Uba4p. Loss of the urmylation pathway components results in sensitivity to a thiol-specific oxidant, as does loss of Ahp1p, implying that urmylation has a role in an oxidative-stress response. Moreover, treatment of cells with thiol-specific oxidants affects the abundance of Ahp1p-Urm1p conjugates. These results suggest that the conjugation of Urm1p to Ahp1p could regulate the function of Ahp1p in antioxidant stress response in Saccharomyces cerevisiae.     

Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.     

Methods for on-chip protein analysis

The unambiguous identification of peptides/proteins is crucial for the definition of the proteome. Using ProteinChip Array technology also known as surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS), we developed experimental protocols and probed test conditions required for the protein identification on ProteinChip surfaces. We were able to directly digest peptides/proteins on-chip surfaces by specific proteases, such as trypsin, and to obtain the peptide mass fingerprint of the sample under investigation by its direct analysis on a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry was performed on several of the resulting tryptic peptides by using collision quadrupole time of flight (Qq-TOF) MS/MS via the ProteinChip interface, thus allowing the unambiguous identification of the protein(s) within the sample. In addition, we were able to identify the C-terminal sequence of peptides by their digestion with carboxypeptidase Y directly on ProteinChip surfaces coupled with SELDI-TOF MS analysis of the resulting peptide mass ladders employing the instrument's protein ladder sequence software. Moreover, the removal of up to nine amino acid residues from the C-terminal end of a peptide extends the functional range of Qq-TOF MS/MS sequence determination to over 3000 m/z. The utility of these procedures for the proteome exploration are discussed.     

Adaptive stress response to menadione-induced oxidative stress in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> KNU5377

The molecular mechanisms involved in the ability of yeast cells to adapt and respond to oxidative stress are of great interest to the pharmaceutical, medical, food, and fermentation industries. In this study, we investigated the time-dependent, cellular redox homeostasis ability to adapt to menadione-induced oxidative stress, using biochemical and proteomic approaches in Saccharomyces cerevisiae KNU5377. Time-dependent cell viability was inversely proportional to endogenous amounts of ROS measured by a fluorescence assay with 2′,7′-dichlorofluorescin diacetate (DCFHDA), and was hypersensitive when cells were exposed to the compound for 60 min. Morphological changes, protein oxidation and lipid peroxidation were also observed. To overcome the unfavorable conditions due to the presence of menadione, yeast cells activated a variety of cell rescue proteins including antioxidant enzymes, molecular chaperones, energy-generating metabolic enzymes, and antioxidant molecules such as trehalose. Thus, these results show that menadione causes ROS generation and high accumulation of cellular ROS levels, which affects cell viability and cell morphology and there is a correlation between resistance to menadione and the high induction of cell rescue proteins after cells enter into this physiological state, which provides a clue about the complex and dynamic stress response in yeast cells.     

Differential analysis of protein expression of Bifidobacterium grown on different carbohydrates

We observed recently that colonic fermentation of lactose might be a major factor in the pathophysiology of lactose intolerance. Proteomic techniques could be helpful in interpreting the metabolic pathways of lactose fermentation. The objective of this study was to explore proteomic methodologies for studying bacterial lactose metabolism that can be used to detect and identify proteins associated with the onset of intolerance symptoms. Differential expression of cytoplasmic proteins of Bifidobacterium animalis, Bifidobacterium breve and Bifidobacterium longum grown on different carbohydrates (lactose, glucose, galactose) was analyzed with surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After fractionation by SDS-PAGE, differentially-expressed proteins were identified with LC-MS/MS. The three strains grown on the same carbohydrate or the same strain grown on glucose or lactose showed differences in SELDI-TOF MS protein profiles. Differences in protein expression were observed in B. breve grown on glucose, galactose or lactose as analyzed with SDS-PAGE. With LC-MS/MS, proteins from Bifidobacterium were identified, which included enzymes for metabolism of lactose, glucose and galactose. In conclusion, the applied techniques can discern differences in protein expression of bacteria metabolizing different carbohydrates. These techniques are promising in studying metabolism of lactose and other substrates in a complex bacterial ecosystem such as the colonic microbiota.     

Proteomic analysis of fast and slow muscles from normal and kyphoscoliotic mice using protein arrays, 2-DE and MS

A proteomic strategy based upon the integrated use of SELDI-TOF/MS, 2-DE and MALDI-TOF/MS has been used to identify a panel of fast muscle protein markers: MLC1F, MLC3F, fast troponin C (STNC) and slow muscle markers: MLC1SB and MLC2v. MLC3F, MLC1F and STNC were virtually absent in the physiologically pure slow soleus muscle of kyphoscoliotic mutant mice compared to control BDmice, whereas MLC2v increased threefold. A SELDI-TOF/MS peak at 18,012 Da in spectra from strong anionic exchange protein array fractions of fast vastus muscle was confirmed as STNC by its specific depletion from crude extracts of vastus muscle using an anti-TNC mAb. SELDI-TOF/MS also identified MLC2F phosphorylation in crude muscle extracts after treatment with alkaline phosphatase. High probability protein identifications were achieved by SELDI-TOF/MS PMF based upon the resolution of large peptides formed by partial cleavage and high peptide coverage. When the pI from 2-D gels and molecular weight estimations from SELDI-TOF/MS were entered into the TagIdent algorithm, high probability protein identity predictions were obtained that were confirmed later by PMF. We confirm that SELDI-TOF/MS can be integrated with other proteomics techniques for the efficient analysis of protein expression changes and PTMs associated with physiological changes in skeletal muscle.     

Differentially expressed proteins in cerulein-stimulated pancreatic acinar cells: implication for acute pancreatitis

The proteins expressed in pancreatic acinar cells during the initiation of acute pancreatitis may determine the severity of the disease. Cerulein pancreatitis is one of the best characterized models for acute pancreatitis. Present study aims to determine the differentially expressed proteins in cerulein-stimulated pancreatic acinar cells as an in vitro model for acute pancreatitis. Rat pancreatic acinar AR42J cells were treated with 10(-8)M cerulein for 12h. The protein patterns separated by two-dimensional electrophoresis using pH gradients of 5-8 were compared between the cells treated without cerulein and those with cerulein. The changed proteins were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. As a result, 10 proteins (Orp150 protein, protein disulfide isomerase related protein, dnaK-type molecular chaperone hsp72-ps1, mitochondrial glutamate dehydrogenase, similar to chaperonin containing TCP-1 beta subunit, RuvB-like protein 1, heterogeneous nuclear ribonucleoprotein H1, aldehyde reductase 1, triosephosphate isomerase 1, peroxiredoxin 2) were up-regulated while four proteins (vasolin-containing protein, 78 kDa glucose-regulated protein precursor, heat shock protein 8, adenosylhomocysteinase) were down-regulated by cerulein in pancreatic acinar AR42J cells. These proteins are related to chaperone, cell defense mechanism against oxidative stress or DNA damage, anti-apoptosis and energy generation. The differentially expressed proteins by ceruein share their functional roles in pancreatic acinar cells, suggesting the possible involvement of oxidative stress, DNA damage, and anti-apoptosis in pathogenesis of acute pancreatitis. Proteins involved in cellular defense mechanism and energy production may protect pancreatic acinar cells during the development of pancreatitis.     

Identification in Saccharomyces cerevisiae of a new stable variant of alkyl hydroperoxide reductase 1 (Ahp1) induced by oxidative stress.

Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern of Saccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point. This form was also present in wild type strains after treatment with tert-butyl hydroperoxide. In vitro enzyme assays showed that Ahp1p had lower specific activity in strains lacking Cu,Zn-SOD. We studied three mutants presenting a reduced production of the low pI variant under oxidative stress conditions. Two of the mutants (C62S and S59D) were totally inactive, thus suggesting that the acidic form of Ahp1p may only appear when the enzyme is functional. The other mutant (S59A) was active in vitro and was more resistant to inactivation by tert-butyl hydroperoxide than the wild type enzyme. Furthermore, the inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress (alkyl hydroperoxide treatment or lack of Cu,Zn-SOD activity but not hydrogen peroxide treatment), the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid (a natural occurring intermediate of the enzymatic reaction) and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive.     

A combination of protein profiling and isotopomer analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometry reveals an active metabolism of the extracellular matrix of 3T3-L1 adipocytes

Differential gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a commonly used protein profiling method. However, observed changes can be explained in multiple ways, one of which is by the protein turnover rate. In order to easily and rapidly obtain information on both the identity and turnover of individual proteins, we applied a combination of protein labeling with L-(ring-2,3,4,5,6 2H5) phenylalanine and MALDI-TOF MS. While the spectrum reveals the identity of a protein, mass isotopomer analysis provides information about the rate of protein labeling as a measure of synthesis or turnover. Using this approach on mature 3T3-L1 adipocytes, we were able to discriminate between rapidly and slowly metabolised proteins. In our isolate, proteins of the cytoskeleton appeared to be slowly metabolised, whereas components of the extracellular matrix, in particular collagen type I alpha 1 (COL1A1) and collagen type I alpha 2 (COL1A2) showed rapid accumulation of newly synthesized proteins. Both proteins appeared to be metabolised in the same ratio as they are present in collagen fibers, i.e. 2:1 (COL1A1: COL1A2). In addition, functionally related proteins were also readily labeled. Taken together, we have shown that a combination of stable isotope labeling and protein profiling by gel electrophoresis and MALDI-TOF analysis can simultaneously provide information on the identity and relative metabolic rate of proteins in eukaryotic cells in a simple, nonhazardous and rapid-throughput way.     

Analysis of human serum proteins by liquid phase isoelectric focusing and matrix-assisted laser desorption/ionization-mass spectrometry

Direct matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of human serum yielded ion signals from only a fraction of the total number of peptides and proteins expected to be in the sample. We increased the number of peptide and protein ion signals observed in the MALDI-TOF mass spectra analysis of human serum by using a prefractionation protocol based on liquid phase isoelectric focusing electrophoresis. This pre-fractionation technique facilitated the MALDI-TOF MS detection of as many as 262 different peptide and protein ion signals from human serum. The results obtained from three replicate fractionation experiments on the same serum sample indicated that 148 different peptide and protein ion signals were reproducibly detected using our isoelectric focusing and MALDI-TOF MS protocol.     

Cellular stress-related protein expression in Helicobacter pylori-infected gastric epithelial AGS cells

Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulceration, and gastric carcinoma. Moreover, H. pylori may induce disease-specific protein expression in gastric epithelial cells. The present study was aimed at determining differentially expressed proteins in H. pylori-infected gastric epithelial AGS cells. AGS cells were treated with H. pylori at a bacterium/cell ratio of 300:1 for 12 h. Altered protein patterns as separated by two-dimensional electrophoresis using pH gradients of 4-7 were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Four differentially expressed proteins, whose expression levels were increased by more than two-fold in H. pylori-infected cells, were analyzed. These proteins (14-3-3 protein alpha/beta, cullin homolog 3, alpha-enolase, ezrin) are known to be related to cell proliferation, cell adhesion, and carcinogenesis, and may be mediated by cellular stress, such as reactive oxygen species. In conclusion, the identification of these differentially expressed proteins provide valuable information for the understanding of the pathophysiologic mechanisms of H. pylori-induced gastric diseases, and may be useful as prognostic indices of H. pylori-related gastric disorders.     

Proteomic analysis of acrylamide gel separated proteins immobilized on polyvinylidene difluoride membranes following proteolytic digestion in the presence of 80% acetonitrile

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry (MS) is a highly accurate and sensitive means of identifying proteins. We have developed a novel method for digesting proteins on polyvinylidene difluoride (PVDF) membranes for subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis. After Tricine sodium dodecyl sulfate (SDS)-PAGE, separated proteins were electroblotted onto PVDF membranes in a semidry discontinuous buffer system, visualized by staining with Coomassie Blue, excised, digested with trypsin or lysC in 80% acetonitrile, and then analyzed by MALDI-TOF MS. This method has several advantages over in-gel digestion in terms of sample handling, sensitivity, and time. We identified 105 fmol of Bacillus subtilis SecA and 100 approximately 500 fmol of standard proteins. We also analyzed the submembrane protein fraction solubilized by 1% n-dodecyl-beta-D-maltoside from B. subtilis membranes after separation by 2-D PAGE, and identified 116 protein spots. This method can detect proteins at the 10 approximately 50 fmol level by pooling more than ten identical electroblotted protein spots.     

A proteomic analysis of cold stress responses in rice seedlings

Using proteomic analysis, an investigation aimed at a better understanding of the molecular adaptation mechanisms of cold stress was carried out in rice (Oryza sativa). The seedlings were exposed to a progressively low temperature stress treatment from normal temperature to 15, 10, and 5 degrees C. Proteins were extracted from the leaves collected from both control and stressed seedlings. By fractionation, approximately 1700 protein spots were separated and visualized on CBB-stained 2-D gels. Sixty protein spots were found to be up-regulated in responding to the progressively low temperature stress and displayed different dynamic patterns. As an initial work, 41 of these proteins were identified using MALDI-TOF MS or ESI/MS/MS. These cold responsive proteins, besides two proteins of unknown function, include four factors of protein biosynthesis, four molecular chaperones, two proteases, and eight enzymes involved in biosynthesis of cell wall components, seven antioxidative/detoxifying enzymes, and proteins linked to energy pathway, as well as a protein involved in signal transduction. The functional proteomes illuminate the facts, at least in plant cell, that protein quality control mediated by chaperones and proteases and enhancement of cell wall components play important roles in tolerance to cold stress. Using TargetP program, the subcellular localization of the identified proteins was analyzed. Proteins (43.9%) were predicted to be located in the chloroplasts, implying that chloroplast proteome is virtually subjective to cold stress. The physiological implications, revealed from the experimental data, are discussed in context of a complex metabolic network in plant cells responsive to cold stress.