Differential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrol

Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardioprotection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apoptosis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and preconditioning- related proteins were identified that were all upregulated by resveratrol: MAPKK, two different alphaB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxiredoxin- 2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -alphaB-crystallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofilament regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling enables the identification of resveratrol induced preconditioning-associated proteins which reflects not only changes in their expression level but also isoforms, post-translational modifications and regulating binding or activating partner proteins.     

Heat-shock protein 27: a potential biomarker for hepatocellular carcinoma identified by serum proteome analysis

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and ranks second in China. The prognosis of HCC remains dismal mainly because of its late diagnosis, especially in patients with coexisting chronic liver diseases. To identify serum biomarkers for HCC, sera from 20 healthy volunteers, 20 hepatitis B virus (HBV) infected patients and 20 HCC patients were selected for screening study and same number of sera into the same three groups were used for validation study. A strategy including sonication, albumin and immunoglobulin G (IgG) depletion and desalting was optimized for screening differentially expressed proteins of low abundance in serum. By 2-DE image analysis and MALDI-TOF-MS/MS identification, eight proteins including heat-shock protein 27 (HSP27), alpha-fetoprotein (AFP), alpha-1 antitrypsin, clusterin, caeruloplasmin, haptoglobin alpha2 chain, tranferrin and transthyretin were found significantly changed among the healthy, HBV and HCC groups. Further validation study by Western blot showed the detection of HSP27 in 90% HCC sera and two HBV sera, but in none of normal sera. Thus, 2-DE based serum proteome analysis can be useful in the screening of serum biomarkers for HCC and HSP27 could aid in the diagnosis of HCC though further validation is needed.     

Proteomic analysis of the cerebrospinal fluid of patients with lumbar disk herniation

To better understand the pathophysiologic mechanisms underlying spinal nerve root injury induced by lumbar disk herniation (LDH), comparative proteomic analysis of cerebrospinal fluid (CSF) between patients with LDH (the experiment group) and the otherwise healthy patients who had had implants removed from healed fractures in the lower limbs (the control group) was carried out using 2-DE followed by LC-IT-MS and database searching. Image analysis of silver-stained 2-DE gels revealed that 15 protein spots showed significant differential expression between the two groups of CSF samples (p < 0.05). After searching the database we found that in CSF of LDH patients, the expression of cystatin C, apolipoprotein A-IV, vitamin D-binding protein, neurofilament triplet L protein, IgG, tetranectin, and hemoglobin were elevated. However, ProSAAS, prostagladin D2 synthase, creatine kinase B, superoxide dismutase 1 and peroxiredoxin 2 were decreased. The subsequent ELISA measured the concentration of tetranectin, vitamin D-binding protein and cystatin C and confirmed the results of proteomic analysis. These identified proteins are involved in the pathophysiological process of spinal nerve root injury caused by herniated lumbar disk. The functional implications of the alterations in the levels of these proteins are discussed in this paper.     

Proteomic analysis in human breast cancer: identification of a characteristic protein expression profile of malignant breast epithelium

Gene expression analysis has become a promising tool in predicting the clinical course of malignant disease and the response to antineoplastic therapy. Surprisingly, only little is known about the protein expression pattern of human tumors. Recent advances in proteomic analysis allow proteins of interest to be identified by their expression and/or modification pattern in 2-DE rather than using the traditional approach of translating gene expression data. To identify a proteomic pattern that is characteristic for malignant breast epithelium, we performed differential 2-DE analysis in sets of microdissected malignant breast epithelia and corresponding adjacent normal breast epithelia from five patients with invasive breast carcinoma. Thirty-two protein spots were found to be selectively regulated in malignant epithelium, and were subjected to MALDI-TOF and/or immunoblotting for protein identification. Thirteen of the identified proteins had previously not been associated with breast cancer. The validity of these findings was confirmed by literature review and immunohistochemistry for identified proteins in an independent cohort of 50 breast cancer specimens. We here describe, for the first time, a proteomic analysis of matched normal and malignant epithelia from invasive breast carcinomas. This strategy leads to a better understanding of oncogenesis at an operational level and helps to characterize the malignant phenotype of individual tumors, and thereby to identify novel targets for antineoplastic therapy.     

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.     

橡胶树死皮病胶乳C-乳清差异表达蛋白质的筛选与鉴定

橡胶树死皮病(Tapping Panel Dryness,TPD)在世界各橡胶种植园普遍发生,给橡胶种植业带来严重的危害。为了更好地了解和阐明死皮病发生、发展的分子机制,本研究应用双向凝胶电泳技术（2-DE）比较橡胶树死皮株与健康株胶乳C-乳清蛋白质组表达的差异。采用固相pH梯度双向凝胶电泳分离橡胶树死皮株与健康株C-乳清的总蛋白质,凝胶经考染显色后,用PDQuest7.40图像分析软件进行比较分析,识别差异表达的蛋白质。这些点经过胶内酶切后进行基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析获取肽质指纹图谱(PMF),Mascot软件搜索SWISS-PROT和NCBInr数据库鉴定蛋白质。结果：①橡胶树死皮株与健康株C-乳清凝胶的平均蛋白质点数分别为1075±35和1134±27,其平均匹配的点数分别为982±38和1008±22,组内图像匹配率达91.89﹪和88.72﹪。②橡胶树死皮株与健康株C-乳清组间的平均匹配蛋白点数为970±25。利用MALDI-TOF-MS质谱技术对40个差异明显的蛋白点进行分析鉴定,通过查询数据库鉴定了27个蛋白质。本研究建立了分辨率高且重复性较好的橡胶树死皮株与 健康株胶乳C-乳清的双向凝胶电泳图谱,并应用质谱技术鉴定了其中表达差异的蛋白质点,这些差异表达的蛋白质可能参与了死皮发生和发展的过程。     

Proteomic profiling of cerebrospinal fluid identifies prostaglandin D2 synthase as a putative biomarker for pediatric medulloblastoma: A pediatric brain tumor consortium study

The aims of this study were to demonstrate the feasibility of centrally collecting and processing high-quality cerebrospinal fluid (CSF) samples for proteomic studies within a multi-center consortium and to identify putative biomarkers for medulloblastoma in CSF. We used 2-DE to investigate the CSF proteome from 33 children with medulloblastoma and compared it against the CSF proteome from 25 age-matched controls. Protein spots were subsequently identified by a combination of in-gel tryptic digestion and MALDI-TOF TOF MS analysis. On average, 160 protein spots were detected by 2-DE and 76 protein spots corresponding to 25 unique proteins were identified using MALDI-TOF. Levels of prostaglandin D2 synthase (PGD2S) were found to be six-fold decreased in the tumor samples versus control samples (p<0.00001). These data were further validated using ELISA. Close examination of PGD2S spots revealed the presence of complex sialylated carbohydrates at residues Asn(78) and Asn(87) . Total PGD2S levels are reduced six-fold in the CSF of children with medulloblastoma most likely representing a host response to the presence of the tumor. In addition, our results demonstrate the feasibility of performing proteomic studies on CSF samples collected from patients at multiple institutions within the consortium setting.     

Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms

Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections.     

Navigated laser capture microdissection as an alternative to direct histological staining for proteomic analysis of brain samples

Proteomic analysis of the brain is complicated by the need to obtain cells from specific anatomical regions, or nuclei. Laser capture microdissection (LCM) is a technique that is precise enough to dissect single cells within a tissue section, and thus could be useful for isolating specific brain nuclei for analysis. However, we and others have previously demonstrated that histological staining protocols used to guide LCM have detrimental effects on protein separation by two-dimensional electrophoresis (2-DE). Here we describe a new LCM method called navigated LCM. This microdissection method uses fixed but unstained tissue as starting material and thus enables us to avoid artifacts induced by tissue staining. By comparing 2-DE results obtained from fixed, unstained LCM brain tissue samples to those obtained from manually dissected samples, we demonstrated that this microdissection process gave similar protein recovery rates and similar resolution of protein spots on 2-DE gels. Moreover, matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis of selected spots from gels derived from control and fixed, LCM samples revealed that the fixation-LCM process had no effect on protein identification. Navigated LCM of tissue sections is therefore a practical and powerful method for performing proteomic studies in specifically defined brain regions.     

Optimization of IPG strip equilibration for the basic membrane protein mABC1

Many proteins with extreme physical properties, including basic and acidic proteins, membrane proteins, and very large proteins, present specific challenges to 2-DE separation. Using a pressure-blotting approach, we demonstrate that a basic integral membrane protein, mitochondrial ATP-binding cassette protein 1 (mABC1), focuses in the IPG strip, but fails to enter into the 2-D SDS-PAGE gel. Through modifying the equilibration conditions between the IPG strip and 2nd dimension, we demonstrate that only by increasing both the volume (from 3 to 6 mL for a 7-cm strip) and SDS concentration (from 2 to 10%) of the equilibration buffer is migration of mABC1 into the 2nd dimension achieved. While 2-DE remains one of the core separation technologies of proteomic analysis, proteins that are extremely basic, hydrophobic, or of large mass present significant challenges to 2-DE separation due to aggregation, oxidation, precipitation, and the physical limitations of the 1-D IPG strip. Since the advent of commercially available IPG strips, numerous groups have experimented with IEF conditions using various detergents alone or in combination, alternative denaturants, and thiol oxidation agents to improve protein focusing. Effective 2-DE separation of membrane proteins has been affected dramatically by these advances in protein solubilization, as well as improvements in isolation of membranes, delipidation, and active in-gel rehydration. Since the development of commercially available basic IPG strips, the most significant advance in the separation of basic proteins has been the introduction of hydroxyethyldisulfides, either alone or in combination with DTT. While hydrophobic proteins were once virtually absent from the 2-D gel, and basic proteins were only visible as dense streaks, now many groups are undertaking large-scale analyses of membranes and basic proteins. Using this base of experimental tools, we embarked on a proteomic analysis of cardiac mitochondrial membranes, hoping to combine the knowledge gained from ongoing targeted protein chemistry and molecular biology studies with a broader-based proteomic analysis. Of particular interest is the inner mitochondrial membrane protein mABC1 (mitochondrial ATP-binding cassette protein 1), which may play a significant role in cardioprotection as part of the mitochondrial ATP-sensitive potassium channels. Therefore, in designing our 2-DE approach, it was crucial to ensure that mABC1 is focused, observable, and quantifiable, despite being an integral membrane protein of pI 9.37.     

Congenital disorder of glycosylation Ia: New differentially expressed proteins identified by 2-DE

Congenital disorders of glycosylation (CDG) comprise a family of inherited multisystemic disorders resulting from the deficiency of glycosylation pathways. N-glycosylation defects are classified as two biochemical and genetic established types, of which CDG-Ia is the most frequent. We performed 2-DE proteomic analysis on serum from two functional hemizygous CDG-Ia patients bearing T237M and D65Y missense changes. Comparative analysis of control/patient serum proteome allowed us to identify differential expression of 14 proteins. The most remarkable groups included proteins involved in immune response, coagulation mechanism and tissue protection against oxidative stress. The patient bearing D65Y mutation had less favourable clinical outcome and showed more abnormalities in the spot patterns, suggesting that the proteomic results might also be correlated with the phenotype of CDG patients. This study describes for the first time the differential expression of α₂-macroglobulin, afamin, fibrin and fibrinogen in CDG disorder and shows how the proteomic approach might be useful for understanding its physiopathology.     

Cypermethrin exposure leads to regulation of proteins expression involved in neoplastic transformation in mouse skin

Cypermethrin, a synthetic pyrethroid insecticide is shown to exert carcinogenic effects in rodents; however, its underlying mechanism remains elusive. Here, we showed the effect of cypermethrin on protein expression involved in neoplastic transformation in mouse skin. Comparative protein expression profiles between untreated control and cypermethrin-treated mouse skin were explored using 2-DE. A total of 27 spots that were statistically significant (p<0.05) and differentially expressed in response to cypermethrin exposure were identified by MALDI-TOF/TOF and LC-MS/MS. Among them, six up-regulated proteins (carbonic anhydrase 3 (Ca 3), Hsp-27, S100A6, galectin-7, S100A9, S100A11) and one down-regulated protein (superoxide dismutase [Cu-Zn] (Sod 1)) are associated with cancer-related key processes. These selected dysregulated proteins were further validated in 2-DE gels of mouse skin treated with known tumorigens (benzo-[a]-pyrene, 12-O-tetradecanoyl-phorbol-13-acetate and mezerein), respectively. Comparative studies showed that Ca 3, S100A6, S100A9, S100A11 and Sod 1 are specific for stages of development and progression of tumors whereas Hsp-27 and galectin-7 are specific for tumor promotion stage by cypermethrin in mouse skin. Furthermore, these chosen proteins confirmed by Western blotting and immunofluorescence staining were consistent with changes in 2-DE check. This proteomic investigation for the first time provides key proteins that will contribute in understanding the mechanism behind cypermethrin-induced neoplastic transformation.     

"Two-in-one" gel for spot matching after two-dimensional electrophoresis

Two-dimensional electrophoresis (2-DE) is one of the most commonly used techniques in proteomic investigations. However, due to the complex interplay of incidence including significant biological sample variations, lengthy steps involved in performing 2-DE as well as exposure time with silver staining, it is sometimes difficult to differentiate authentic differences caused by drug treatment with those artifacts caused by sample variations, running conditions of 2-DE as well as treatment time in silver staining etc. If we can compare pooled samples of control and treatment groups run in a single gel and stained together, we would be more comfortable with our findings. We propose here a low cost and highly effective method for locating differentially expressed proteins before and after drug treatment. This "two-in-one gel" technique might partially solve the problems mentioned above.     

果实蛋白质组学研究的实验方法

双向电泳技术是蛋白质组学研究的基本方法之一。果实由于富含糖、多酚、单宁和有机酸等物质,蛋白质的提取比其它植物组织更加困难。本文主要介绍不同果实蛋白质的提取、等电聚焦系统和凝胶染色技术,并建立了一套适用于桃、樱桃、苹果、芒果和冬枣等多种果实蛋白质组学的研究方法。结果表明,采用匀浆法和酚抽提法提取果实的蛋白质,裂解缓冲液2溶解蛋白质,并用固相pH梯度进行等电聚焦,可以获得背景清晰和分辨率高的凝胶图谱,具有较好的重复性,可用于果实蛋白质组学的研究。我们的研究结果显示,固相干胶条与IEF管胶相比,具有更加明显的优势。而不同的染色方法,对结果影响不大。     

Comprehensive quantitative proteome analysis of 20S proteasome subtypes from rat liver by isotope coded affinity tag and 2-D gel-based approaches

Quantitative protein profiling is an essential part of proteomics and requires technologies that accurately, reproducibly, and comprehensively identify and quantify proteins. Over the past years, many quantitative proteomic methods have been developed. Here, 20S proteasome subtypes isolated from rat were compared by four approaches based on the combination of isotope-coded affinity tag (ICAT), 2-DE, LC and ESI and MALDI MS: (i) 2-DE, (ii) ICAT/2-DE MALDI-MS, (iii) ICAT/LC-ESI-MS, (iv) ICAT/LC-MALDI-MS. A definite qualitative advantage of 2-DE gels was the separation of all known protein species, the identification of cysteine sulfoxide of alpha-4 (RC6-IS) and N-terminal acetylation of several subunits. Furthermore, quantitative differences between the standard subunits beta-2, and beta-5 and their immunosubunits were only detected by 2-DE image analysis revealing a higher replacement of standard- by immuno-beta-subunits in subtype IV. It was obvious that for relative quantification only protein spot and mass peaks with a certain level of intensity displayed acceptable values of SD. However, ICAT in conjunction with LC/MALDI-MS was the most accurate method for quantification. The experimental data of this investigation are accessible via http://www.mpiib-berlin.mpg.de/2D-PAGE/.     

Primary cell cultures arising from normal kidney and renal cell carcinoma retain the proteomic profile of corresponding tissues

Renal cell carcinoma (RCC) tissue is composed of a mixture of neoplastic and normal cells, which complicate proteome analysis. The aim of our study was to investigate whether it is feasible to establish primary cell cultures of RCC and of renal cortex maintaining the tissue phenotype along with a more homogeneous and enriched cytological material. Fourteen (82.3%) primary cultures from 17 surgical cases were established and characterized by morphology, growth rate, immunocytochemistry, and molecular analysis performed by Real-time PCR, Western blotting, two-dimensional electrophoresis (2-DE), and mass spectrometry. Cultures showed >90% cytokeratine-positive epithelial cells. In primary tumor cultures, the molecular phenotype of manganese superoxide dismutase and heat shock protein 27 was the same as that found in tumor tissues with overexpression and increased number of isoforms. Moreover, 27 out 28 specific proteins and their isoforms, present in spots excised from 2-DE gel of cortex or RCC cultures, corresponded to those identified on the 2-DE tissue cortex reference map, suggesting that these primary cultures retain the proteomic profile of the corresponding tissues.     

Plasma proteome analysis in diet-induced obesity-prone and obesity-resistant rats

One of the major issues in the field of obesity is why some humans become obese and others resist development of obesity when exposed to high-calorie diets. Despite the same genetic background, namely obesity-prone (OP) and -resistant (OR) rats, differing responses have been demonstrated in a high fat diet-induced rodent model. The aim of the present study was to discover novel obesity-related biomarkers for susceptibility and/or resistance to obesity by proteomic analysis of OP and OR rat plasma. After feeding of high fat diet, OP rats gained approximately 25% more body weight than OR rats and were used for proteomic analysis using 2-DE combined with MALDI-TOF-MS. We categorized identified proteins into three groups by analysis of both average spot density in each group and individual spot density of six rats as a function of body weight. Consequently, category (1) included inter-α-inhibitor H4 heavy chain and fetuin B precursor, which can be used as novel plasma biomarkers for risk of obesity. Nine proteins of category (2) and (3) can also be plausible plasma markers in the study of obesity. This proteomic study is an important advancement over the previous steps needed for identification of OP and OR rats.     

Premature termination codon at the sterol 27-hydroxylase gene causes cerebrotendinous xanthomatosis in a French family

Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive lipid-storage disease caused by mutations in the sterol 27-hydroxylase gene (CYP27). So far several mutations causing CTX have been identified and characterized. A new mutation creating an insertion of cytosine at position 6 in the cDNA, which is expected to result in a frameshift and a premature termination codon at codon 179, has been identified in a French family. The mutation creates a new site for the restriction endonuclease HaeIII.     

Proteomic analysis for inhibitory effect of chitosan oligosaccharides on 3T3-L1 adipocyte differentiation

In the present study, we performed a differential proteomic analysis using 2-DE combined with MS to clarify the molecular mechanism for the suppressive effect of chitosan oligosaccharides (CO) during differentiation of adipocyte 3T3-L1. Cell differentiation was significantly inhibited by CO at the concentration of 4 mg/mL. Protein mapping of adipocyte homogenates by 2-DE revealed that numerous protein spots were differentially altered in response to CO treatment. Out of 50 identified proteins showing significant alterations, six were up-regulated and 44 were down-regulated by CO treatment in comparison to control mature adipocytes. Among them, most of the proteins are associated with lipid metabolism, cytoskeleton, and redox regulation, in which the levels of farnesyl diphosphate synthetase (FDS), dedicator of cytokinesis 9 (DOCK9), and chloride intracellular channel 1 (CLIC1) were significantly reduced (>two-fold) with CO treatment. These results have not previously been examined in the context of adipogenesis, and thus can be used as novel biomarkers. Taken together with immunoblot analysis, it was concluded that the inhibitory effect of CO on adipocyte differentiation was mediated by C/EBPalpha and PPARgamma pathway through significant downregulations of important adipogenic molecules such as fatty acid binding protein and glucose transporter 4.     

Proteomic changes in the pressure overloaded right ventricle after 6 weeks in young rats: correlations with the degree of hypertrophy

Right ventricular (RV) hypertrophy is an important problem in congenital heart disease. We determined the alterations in phenotype that occur in the initial phase of RV hypertrophy and their possible correlations with the degree of hypertrophy. Therefore, we performed a differential proteomic profiling study on RV hypertrophy using an animal model of pulmonary artery banding (PAB) in parallel with hemodynamic characterization. The RV homogenates were subfractionated in myofilament and cytoplasmic proteins, which subsequently were separated by two-dimensional gel electrophoresis (2-DE), excised, and analyzed by mass spectrometry (MS). The cytoplasmic fraction showed expression changes in metabolic proteins, indicative of a shift from fatty acid to glucose as a substrate for energy supply. Up-regulation of three HSP-27s (1.9-, 1.7-, and 3.5-fold) indicated an altered stress response in RV hypertrophy. Detailed analysis by immunoblotting and MS showed that two of these HSP-27s were at least phosphorylated on Ser15. The myofilament fraction showed up-regulation of desmin and alpha-B-crystallin (1.4-and 1.3-fold, respectively). This alteration in desmin was confirmed by 1-DE immunoblots. Certain differentially expressed proteins, such as HSP-27, showed a significant correlation with the RV weight to the body weight ratio in the PAB rats, suggesting an association with the degree of hypertrophy.