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Background: Marine medaka is among the most popular models of fish species for ecotoxicology and environmental research and proteomic studies are useful tools for understanding the molecular responses of medaka upon exposure to different environmental stressors. The preparation of high-quality protein samples is the key to producing high-quality two-dimensional gel electrophoresis (2-DE) results for proteomic analysis. In recent years, Trizol-based protein extraction has been gaining popularity because of its promising performance in producing high-quality 2-DE as well as the convenience of the method. Methods: Three Trizol-based approaches (Trizol method, Aliquot Trizol method and Trizol method with a commercial clean-up kit) were used to extract proteins from a marine medaka sample and 2-DE profiles were produced. Quality of the 2-DE profiles and effectiveness of the extraction methods were evaluated. For comparison, two common protein extraction methods (lysis buffer method and trichloroacetic acid (TCA)/acetone precipitation extraction) were also applied in parallel to Trizol-based approaches. Results: Any of the three Trizol-based approaches produced a high-quality 2-DE profile of marine medaka compared with both lysis buffer method and TCA/acetone precipitation extraction. In addition, Trizol method with a commercial clean-up kit produced the best 2-DE profile in terms of background clarity, number of spots and resolution of proteins. Conclusions: Trizol-based approaches offered better choices than traditional protein extraction methods for 2-DE analysis of marine medaka. The modified version of Trizol method with a commercial clean-up kit was shown to produce the best 2-DE profile. [ABSTRACT FROM AUTHOR]

Background: Traditional studies of the cardiac proteome have mainly investigated in an animal model by two-dimensional gel electrophoresis (2-DE). However, the results have not been of satisfactory quality for an understanding of the underlying mechanism. Recent quantitative proteomic methods have been improved to overcome these limitations. To comprehensively study the cardiac proteome in a rat model of ischemia-reperfusion (IR), we developed a tandem mass tag (TMT)-based quantitative proteomic strategy. Furthermore, using this strategy, we examined the molecular mechanisms underlying the prevention of myocardial infarction by the intake of Triticum aestivum L. extract (TALE), a representative dietary fiber grain. Methods: Cardiac proteomes were analyzed by 2-DE as a gel-based approach, and TMT labeling coupled with two-dimensional liquid chromatography (2D-LC) and tandem mass spectrometry (MS/MS) as a non-gel-based quantitative approach. Additionally, gene ontology annotation was conducted by PANTHER database. Several proteins of interest were verified by a Western blot analysis. Results: Total 641 proteins were identified commonly from two independent MS datasets using 2D-LC MS/MS. Among these, we identified 151 IR-related proteins that were differentially expressed between the sham-operation group and IR group, comprising 62 up-regulated proteins and 89 down-regulated proteins. Most of the reduced proteins were involved in metabolic processes. In addition, 57 of the IR-related proteins were affected by TALE intake, representing 25 up-regulated proteins and 32 down-regulated proteins. In particular, TALE intake leads to a switch in metabolism to reduce the loss of high-energy phosphates and the accumulation of harmful catabolites (especially reactive oxygen species (ROS)) and to maintain cytoskeleton balance, leading to a reduction in cardiac IR injury. Conclusions: Our study provides a comprehensive proteome map of IR-related proteins and potential target proteins and identifies mechanisms implicated in the prevention of myocardial infarction by TALE intake in a rat IR model. [ABSTRACT FROM AUTHOR]

Abstract Background Alfalfa (Medicago sativa) is the most extensively cultivated forage legume in the world, and salinity stress is the most problematic environmental factors limiting alfalfa production. To evaluate alfalfa tissue variations in response to salt stress, comparative physiological and proteomic analyses were made of salt responses in the roots and shoots of the alfalfa. Method A two-dimensional gel electrophoresis (2-DE)-based proteomic technique was employed to identify the differentially abundant proteins (DAPs) from salt-treated alfalfa roots and shoots of the salt tolerance cultivars Zhongmu No 1 cultivar, which was subjected to a range of salt stress concentrations for 9 days. In parallel, REL, MAD and H2O2 contents, and the activities of antioxidant enzymes of shoots and roots were determinand. Result Twenty-seven spots in the shoots and 36 spots in the roots that exhibited showed significant abundance variations were identified by MALDI-TOF-TOF MS. These DAPs are mainly involved in the biological processes of photosynthesis, stress and defense, carbohydrate and energy metabolism, second metabolism, protein metabolism, transcriptional regulation, cell wall and cytoskeleton metabolism, ion transpor, signal transduction. In parallel, physiological data were correlated well with our proteomic results. It is worth emphasizing that some novel salt-responsive proteins were identified, such as CP12, pathogenesis-related protein 2, harvest-induced protein, isoliquiritigenin 2′-O-methyltransferase. qRT-PCR was used to study the gene expression levels of the four above-mentioned proteins; four patterns are consistent with those of induced protein. Conclusion The primary mechanisms underlying the ability of alfalfa seedlings to tolerate salt stress were photosynthesis, detoxifying and antioxidant, secondary metabolism, and ion transport. And it also suggests that the different tissues responded to salt-stress in different ways.

Background: Somatic embryogenesis is a complex process regulated by numerous factors. The identification of proteins that are differentially expressed during plant development could result in the development of molecular markers of plant metabolism and provide information contributing to the monitoring and understanding of different biological responses. In addition, the identification of molecular markers could lead to the optimization of protocols allowing the use of biotechnology for papaya propagation and reproduction. This work aimed to investigate the effects of polyethylene glycol (PEG) on somatic embryo development and the protein expression profile during somatic embryo maturation in papaya (Carica papaya L.). Results: The maturation treatment supplemented with 6% PEG (PEG6) resulted in the greatest number of somatic embryos and induced differential protein expression compared with cultures grown under the control treatment. Among 135 spots selected for MS/MS analysis, 76 spots were successfully identified, 38 of which were common to both treatments, while 14 spots were unique to the control treatment, and 24 spots were unique to the PEG6 treatment. The identified proteins were assigned to seven categories or were unclassified. The most representative class of proteins observed in the control treatment was associated with the stress response (25.8%), while those under PEG6 treatment were carbohydrate and energy metabolism (18.4%) and the stress response (18.4%). Conclusions: The differential expression of three proteins (enolase, esterase and ADH3) induced by PEG6 treatment could play an important role in maturation, and these proteins could be characterized as candidate biomarkers of somatic embryogenesis in papaya. [ABSTRACT FROM AUTHOR]

Background: Fusarium wilt of banana is one of the most destructive diseases in the world. This disease has caused heavy losses in major banana production areas. Except for molecular breeding methods based on plant defense mechanisms, effective methods to control the disease are still lacking. Dynamic changes in defense mechanisms between susceptible, moderately resistant, and highly resistant banana and Fusarium oxysporum f. sp. cubense tropical race 4 (Foc4) at the protein level remain unknown. This research reports the proteomic profile of three banana cultivars in response to Foc4 and transcriptional levels correlated with their sequences for the design of disease control strategies by molecular breeding. Results: Thirty-eight differentially expressed proteins were identified to function in cell metabolism. Most of these proteins were positively regulated after Foc4 inoculation. These differentially regulated proteins were found to have important functions in banana defense response. Functional categories implicated that these proteins were associated with pathogenesis-related (PR) response; isoflavonoid, flavonoid, and anthocyanin syntheses; cell wall strengthening; cell polarization; reactive oxygen species production and scavenging; jasmonic acid-, abscisic acid-, and auxin-mediated signaling conduction; molecular chaperones; energy; and primary metabolism. By comparing the protein profiles of resistant and susceptible banana cultivars, many proteins showed obvious distinction in their defense mechanism functions. PR proteins in susceptible 'Brazil' were mainly involved in defense. The proteins related to PR response, cell wall strengthening and antifungal compound synthesis in moderately resistant 'Nongke No.1' were mainly involved in defense. The proteins related to PR response, cell wall strengthening, and antifungal compound synthesis in highly resistant 'Yueyoukang I' were mainly involved in defense. 12 differentially regulated genes were selected to validate through quantitative real time PCR method. Quantitative RT-PCR analyses of these selected genes corroborate with their respective protein abundance after pathogen infection. Conclusions: This report is the first to use proteomic profiling to study the molecular mechanism of banana roots infected with Foc4. The differentially regulated proteins involved in different defense pathways are likely associated with different resistant levels of the three banana cultivars. [ABSTRACT FROM AUTHOR]

Hepatic fibrosis is an inevitable process in the progression of chronic HBV infection to hepatic cirrhosis, but its detailed mechanism is still unknown. Clinic serum biomarkers of HBV hepatic cirrhosis were scanned by proteomic methods. We used two-dimensional electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) to separate and identify the proteins which were differentially expressed in the serum of patients with hepatic fibrosis compared to HBV carriers. We identified 27 differentially expressed proteins, of which 19 proteins were up-regulated and 8 proteins were down-regulated in the serum of patients with hepatic fibrosis compared to HBV carriers. The expression level of enolase-1 (α-enolase) was decreased while the level of thrombospondin-1 (TSP-1) increased in the serum of patients with hepatic fibrosis by western blot. Enolase-1 and TSP-1 may be useful as biomarkers for the clinic diagnosis of hepatic fibrosis, but further study is necessary. [ABSTRACT FROM AUTHOR]

Background: This study examined the effect of storage temperature on the protein profile of human plasma. Plasma samples were stored for 13 days at -80°C, -20°C, +4°C and room temperature (20-25°C) prior to proteomic analysis. The proteomic comparisons were based on the differences of mean intensity values of protein spots between fresh plasma samples (named "time zero") and plasma samples stored at different temperatures. To better understand the thermally induced biochemical changes that may affect plasma proteins during storage we identified proteins with different expressions with respect to the time zero sample. Results: Using two-dimensional electrophoresis followed by MALDI-TOF MS and /or LC-MS/MS 20 protein spots representing 10 proteins were identified with significant differences in abundance when stored at different temperatures. Our results, in agreement with various authors, indicate that during storage for a short period (13 days) at four different temperatures plasma proteins were more affected by degradation processes at +4°C compared to the other temperatures analysed. However, we founded that numerous protein spots (vitamin D binding protein, alpha-1-antitrypsin, serotransferrin, apoplipoprotein A-I, apolipoprotein E, haptoglobin and complement factor B) decrease in abundance with increasing temperature up to 4°C, but at room temperature their intensity mean values are similar to those of time zero and -80°C. We hypothesize that these proteins are labile at 4°C, but at the same time they are stable at room temperature (20-25°C). Furthermore we have grouped the proteins based on their different sensitivity to the storage temperature. Spots of serum albumin, fibrinogen gamma chain and haptoglobin are more resistant to the higher temperatures tested, as they have undergone changes in abundance only at room temperature; conversely, other spots of serum albumin, fibrinogen beta chain and serotransferrin are more labile as they have undergone changes in abundance at all temperatures except at -80°C. Conclusions: Although there are many studies concerning protein stability of clinical samples during storage these findings may help to provide a better understanding of the changes of proteins induced by storage temperature. [ABSTRACT FROM AUTHOR]

Background: Gel-based proteomic is a popular and versatile method of global protein separation and quantification. However, separation of basic protein still represents technical challenges with recurrent problems of resolution and reproducibility. Results: Three different protocols of protein loading were compared using MCF7 cells proteins. In-gel rehydration, cup-loading and paper-bridge loading were first compared using 6-11 IPG strips, as attempted, in-gel rehydration gave large horizontal steaking; paper-bridge loading displayed an interesting spot resolution, but with a predominant loss of material; cup-loading was selected as the most relevant method, but still needing improvement. Twelve cup-loading protocols were compared with various strip rehydration, and cathodic wick solutions. Destreak appeared as better than DTT for strip rehydration; the use of isopropanol gave no improvement. The best 2DE separation was observed with cathodic wicks filled with rehydration solution complemented with DTT. Paper-bridge loading was finally analyzed using non-limited samples, such as bovine milk. In this case, new spots of basic milk proteins were observed, with or without paper wicks. Conclusion: According to this technical study of basic protein focalization with IPG strips, the cup-loading protocol clearly displayed the best resolution and reproducibility: strips were first rehydrated with standard solution, then proteins were cup-loaded with destreak reagent, and focalisation was performed with cathodic wicks filled with rehydration solution and DTT. Paper-bridge loading could be as well used, but preferentially with non-limited samples. [ABSTRACT FROM AUTHOR]

The article presents information on a study conducted to optimize the expression of proteins on 2-dimensional gel electrophoresis (2-DE) gels to develop a reference proteome for non-osteoarthritic knee synovial fluid (SF). In the study, 3 samples were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit before 2-DE analysis. The study concluded that 2-D Clean-Up Kit treated SF samples showed better results.

The article presents information on an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers of Down Syndrome (DS) in maternal blood plasma. In the study, maternal plasma samples were obtained from mothers carrying DS affected fetuses. The study concluded that despite the in depth two-dimensional gel electrophoresis (2DE) approach, the results showed deficiencies of gel-based proteomics for detection of plasma biomarkers.

The article presents information on a study, in which a proteomic technique was used to determine the effects of Phytophthora sojae infection on soybean hypocotyls. In the study, 46 differentially expressed proteins were identified in soybean hypocotyls infected with P. sojae, by using two-dimensional electrophoresis and matrix-assisted laser desorption-ionization.

The article presents information on a study conducted on the differential effects of a post-anthesis fertilizer on the wheat flour proteome. In the study, plants of Triticum aestivum were grown with or without post-anthesis fertilization (PAF), and quantitative 2-dimensional gel electrophoresis (2-DE) was used to analyze protein composition of the resulting flour. The study concluded that A 2-DE approach revealed changes in the wheat flour proteome due to PAF.

Background: High temperature is a critical abiotic stress that reduces crop yield and quality. Rice (Oryza sativa L.) plants remodel their proteomes in response to high temperature stress. Moreover, phosphorylation is the most common form of protein post-translational modification (PTM). However, the differential expression of phosphoproteins induced by heat in rice remains unexplored. Methods: Phosphoprotein in the leaves of rice under heat stress were displayed using two-dimensional electrophoresis (2-DE) and Pro-Q Diamond dye. Differentially expressed phosphoproteins were identified by MALDITOF- TOF-MS/MS and confirmed by Western blotting. Results: Ten heat-phosphoproteins were identified from twelve protein spots, including ribulose bisphos-phate carboxylase large chain, 2-Cys peroxiredoxin BAS1, putative mRNA binding protein, Os01g0791600 protein, OSJNBa0076N16.12 protein, putative H(+)-transporting ATP synthase, ATP synthase subunit beta and three putative uncharacterized proteins. The identification of ATP synthase subunit beta was further validated by Western-blotting. Four phosphorylation site predictors were also used to predict the phosphorylation sites and the specific kinases for these 10 phosphoproteins. Conclusion: Heat stress induced the dephosphorylation of RuBisCo and the phosphorylation of ATP-b, which decreased the activities of RuBisCo and ATP synthase. The observed dephosphorylation of the mRNA binding protein and 2-Cys peroxiredoxin may be involved in the transduction of heat-stress signaling, but the functional importance of other phosphoproteins, such as H+-ATPase, remains unknown. [ABSTRACT FROM AUTHOR]

Background: Actinobacillus pleuropneumoniae (APP) is one of the most important swine pathogens worldwide. Identification and characterization of novel antigenic APP vaccine candidates are underway. In the present study, we use an immunoproteomic approach to identify APP protein antigens that may elicit an immune response in serotype 1 naturally infected swine and serotype 1 virulent strain S259-immunized rabbits. Results: Proteins from total cell lysates of serotype 1 APP were separated by two-dimensional electrophoresis (2DE). Western blot analysis revealed 21 immunoreactive protein spots separated in the pH 4-7 range and 4 spots in the pH 7-11 range with the convalescent sera from swine; we found 5 immunoreactive protein spots that separated in the pH 4-7 range and 2 in the pH 7-11 range with hyperimmune sera from S259-immunized rabbits. The proteins included the known antigens ApxIIA, protective surface antigen D15, outer membrane proteins P5, subunit NqrA. The remaining antigens are being reported as immunoreactive proteins in APP for the first time, to our knowledge. Conclusions: We identified a total of 42 immunoreactive proteins of the APP serotype 1 virulent strain S259 which represented 32 different proteins, including some novel immunoreactive factors which could be researched as vaccine candidates. [ABSTRACT FROM AUTHOR]

Background: Bladder transitional cell carcinoma (BTCC) is the fourth most frequent neoplasia in men, clinically characterized by high recurrent rates and poor prognosis. Availability of urinary tumor biomarkers represents a convenient alternative for early detection and disease surveillance because of its direct contact with the tumor and sample accessibility. Results: We tested urine samples from healthy volunteers and patients with low malignant or aggressive BTCC to identify potential biomarkers for early detection of BTCC by two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) and bioinformatics analysis. We observed increased expression of five proteins, including fibrinogen (Fb), lactate dehydrogenase B (LDHB), apolipoprotein-A1 (Apo-A1), clusterin (CLU) and haptoglobin (Hp), which were increased in urine samples of patients with low malignant or aggressive bladder cancer. Further analysis of urine samples of aggressive BTCC showed significant increase in Apo-A1 expression compared to low malignant BTCC. Apo-A1 level was measured quantitatively using enzyme-linked immunosorbent assay (ELISA) and was suggested to provide diagnostic utility to distinguish patients with bladder cancer from controls at 18.22 ng/ml, and distinguish patients with low malignant BTCC from patients with aggressive BTCC in two-tie grading system at 29.86 ng/ml respectively. Further validation assay showed that Apo-A1 could be used as a biomarker to diagnosis BTCC with a sensitivity and specificity of 91.6% and 85.7% respectively, and classify BTCC in two-tie grading system with a sensitivity and specificity of 83.7% and 89.7% respectively. Conclusion: Taken together, our findings suggest Apo-A1 could be a potential biomarker related with early diagnosis and classification in two-tie grading system for bladder cancer. [ABSTRACT FROM AUTHOR]

Background: Unravelling the serum proteome is the subject of intensified research. In this regard, two-dimensional electrophoresis coupled with MALDI MS analysis is still one of the most commonly used method. Despite some improvements, there is the need for better protocols to enable comprehensive identification of serum proteins. Here we report a combination of two proteomic strategies, zoom in acidic and neutral part of 2-D gels and an application of two optimised matrix preparations for MALDI-MS analyses to simplify serum proteome mapping. Results: Mouse serum proteins were separated by 2-D electrophoresis at the pH ranges 3-10 and 4-7, respectively. Then in gel tryptic digests were analysed by MALDI-MS. Notably, sample-matrix preparations consisted of either a thin-layer α-ciano-4-hydroxycinnamic acid (CHCA) matrix deposition or a matrix-layer 2,5-dihydroxybenzoic acid (DHB). This enabled an identification of 90 proteins. The herein reported method enhanced identification of proteins by 32% when compared with previously published studies of mouse serum proteins, using the same approaches. Furthermore, experimental improvements of matrix preparations enabled automatic identification of mouse proteins, even when one of the two matrices failed. Conclusion: We report a simple and reliable protocol for serum proteome analysis that combines an optimized resolution of 2-D gels spots and improved sample-matrix preparations for MALDI-MS analysis. The protocol allowed automated data acquisition for both CHCA and DHB and simplified the MS data acquisition therefore avoiding time-consuming procedures. The simplicity and reliability of the developed protocol may be applied universally. [ABSTRACT FROM AUTHOR]

Background: Trypanosoma cruzi, a flagellate protozoan, is the etiological agent of Chagas disease, a chronic illness that causes irreversible damage to heart and digestive tract in humans. Previous 2-DE analyses of T. cruzi proteome have not focused on basic proteins, possibly because of inherent difficulties for optimizing 2-DE in the alkaline pH range. However, T. cruzi wide pH range 2-DE gels have shown few visible spots in the alkaline region, indicating that the parasite either did not have an appreciable amount of alkaline proteins or that these proteins were underrepresented in the 2-DE gels. Results: Different IEF conditions using 6-11 pH gradient strips were tested for separation of T. cruzi alkaline proteins. The optimized methodology described here was performed using anodic "paper bridge" sample loading supplemented by increased concentration of DTT and Triton X-100 on Multiphor II (GE Healthcare) equipment and an electrode pad embedded in DTT- containing solution near the cathode in order to avoid depletion of reducing agent during IEF. Landmark proteins were identified by peptide mass fingerprinting allowing the production of an epimastigote 2-DE map. Most identified proteins corresponded to metabolic enzymes, especially those related to amino acid metabolism. The optimized 2- DE protocol was applied in combination with the "two-in-one gel" method to verify the relative expression of the identified proteins between samples from epimastigote and trypomastigote life stages. Conclusion: High resolution 2-DE gels of T. cruzi life forms were achieved using the optimized methodology and a partial epimastigote alkaline 2-DE map was built. Among 700 protein spots detected, 422 were alkaline with a pI above 7.0. The "two-in-one gel" method simplified the comparative analysis between T. cruzi life stages since it minimized variations in spot migration and silver-stained spot volumes. The comparative data were in agreement with biological traits of T. cruzi life forms and also corroborated previous T. cruzi proteomic studies. For instance, enzymes related to amino acid metabolism and dehydrogenases were more abundant in epimastigote 2-DE gel whilst trans-sialidase and a paraflagellar protein were found specifically in the trypomastigote 2-DE profile. [ABSTRACT FROM AUTHOR]

Abstract Background Gel-based proteomic is a popular and versatile method of global protein separation and quantification. However, separation of basic protein still represents technical challenges with recurrent problems of resolution and reproducibility. Results Three different protocols of protein loading were compared using MCF7 cells proteins. In-gel rehydration, cup-loading and paper-bridge loading were first compared using 6–11 IPG strips, as attempted, in-gel rehydration gave large horizontal steaking; paper-bridge loading displayed an interesting spot resolution, but with a predominant loss of material; cup-loading was selected as the most relevant method, but still needing improvement. Twelve cup-loading protocols were compared with various strip rehydration, and cathodic wick solutions. Destreak appeared as better than DTT for strip rehydration; the use of isopropanol gave no improvement. The best 2DE separation was observed with cathodic wicks filled with rehydration solution complemented with DTT. Paper-bridge loading was finally analyzed using non-limited samples, such as bovine milk. In this case, new spots of basic milk proteins were observed, with or without paper wicks. Conclusion According to this technical study of basic protein focalization with IPG strips, the cup-loading protocol clearly displayed the best resolution and reproducibility: strips were first rehydrated with standard solution, then proteins were cup-loaded with destreak reagent, and focalisation was performed with cathodic wicks filled with rehydration solution and DTT. Paper-bridge loading could be as well used, but preferentially with non-limited samples.

Background: Alfalfa ( Medicago sativa ) is the most extensively cultivated forage legume in the world, and salinity stress is the most problematic environmental factors limiting alfalfa production. To evaluate alfalfa tissue variations in response to salt stress, comparative physiological and proteomic analyses were made of salt responses in the roots and shoots of the alfalfa., Method: A two-dimensional gel electrophoresis (2-DE)-based proteomic technique was employed to identify the differentially abundant proteins (DAPs) from salt-treated alfalfa roots and shoots of the salt tolerance cultivars Zhongmu No 1 cultivar, which was subjected to a range of salt stress concentrations for 9 days. In parallel, REL, MAD and H 2 O 2 contents, and the activities of antioxidant enzymes of shoots and roots were determinand., Result: Twenty-seven spots in the shoots and 36 spots in the roots that exhibited showed significant abundance variations were identified by MALDI-TOF-TOF MS. These DAPs are mainly involved in the biological processes of photosynthesis, stress and defense, carbohydrate and energy metabolism, second metabolism, protein metabolism, transcriptional regulation, cell wall and cytoskeleton metabolism, ion transpor, signal transduction. In parallel, physiological data were correlated well with our proteomic results. It is worth emphasizing that some novel salt-responsive proteins were identified, such as CP12, pathogenesis-related protein 2, harvest-induced protein, isoliquiritigenin 2'-O-methyltransferase. qRT-PCR was used to study the gene expression levels of the four above-mentioned proteins; four patterns are consistent with those of induced protein., Conclusion: The primary mechanisms underlying the ability of alfalfa seedlings to tolerate salt stress were photosynthesis, detoxifying and antioxidant, secondary metabolism, and ion transport. And it also suggests that the different tissues responded to salt-stress in different ways.

A correction to the article "A Comparison of Imputation Procedures and Statistical Tests for the Analysis of Two-Dimensional Electrophoresis Data," published in the previous issue of the journal is presented.

Background: Somatic embryogenesis is a complex process regulated by numerous factors. The identification of proteins that are differentially expressed during plant development could result in the development of molecular markers of plant metabolism and provide information contributing to the monitoring and understanding of different biological responses. In addition, the identification of molecular markers could lead to the optimization of protocols allowing the use of biotechnology for papaya propagation and reproduction. This work aimed to investigate the effects of polyethylene glycol (PEG) on somatic embryo development and the protein expression profile during somatic embryo maturation in papaya (Carica papaya L.)., Results: The maturation treatment supplemented with 6% PEG (PEG6) resulted in the greatest number of somatic embryos and induced differential protein expression compared with cultures grown under the control treatment. Among 135 spots selected for MS/MS analysis, 76 spots were successfully identified, 38 of which were common to both treatments, while 14 spots were unique to the control treatment, and 24 spots were unique to the PEG6 treatment. The identified proteins were assigned to seven categories or were unclassified. The most representative class of proteins observed in the control treatment was associated with the stress response (25.8%), while those under PEG6 treatment were carbohydrate and energy metabolism (18.4%) and the stress response (18.4%)., Conclusions: The differential expression of three proteins (enolase, esterase and ADH3) induced by PEG6 treatment could play an important role in maturation, and these proteins could be characterized as candidate biomarkers of somatic embryogenesis in papaya.