Cover Picture: Proteomics 5/2009

In this issue of Proteomics you will find the following highlighted articles: Heart (pump) broken? Hearts are pumps within pumps within channels and pumps. Calcium is pumped, potassium, sodium, amino acids, and electrons are all pumped, channeled or driven until, finally, blood is pumped. Failure of one or more pumps leads to a heart attack. This report from Zlatkovic et al. looks at the sub‐proteome associated with hypertensive failure of the K⁺ATP channel and associated cardiomyopathy that develops in KIR6.2 knock‐out mice. Out of >900 reproducible 2‐DE spots, 81 displayed significant over‐ or under‐expression, a number of which validated previously proposed interactions with the Kir6.2 channel. Two‐thirds were down‐regulations, including creatine kinase, adenylate kinase, and lactate dehydrogenase. A total of 114 proteins were ontologically mapped into the K⁺ATP‐dependent sub‐proteome and a role in hypertensive heart failure. Interaction mapping found >240 nodes and >1200 interactions/edges. A good foundation for future work. Zlatkovic, J. et al., Proteomics 2009, 9, 1314‐1325. The deeper you dig, the more you find A classical biochemist interested in protein‐protein interactions purifies his protein away from other proteins, seeking the highest “‐fold purification”. A proteomicist, on the other hand, looks for “consistent contamination” – i.e. association – of the protein of interest with other proteins. This requires high resolution separations and high accuracy concentration determinations. You can only work with species with concentrations above the detection limit (DL) for the detection method. 2‐DE MS has a DL of approximately 10^?8 M, LC‐MS/MS is ～10^?10 M and saturating Cy5 dye method is ～10^?13 M. Archakov et al. report on an atomic force microscope technique that can yield a DL of 10^?16 M when the target is irreversibly fixed to the bait to avoid the losses due to dissociation kinetics. At that level, over 1 000 000 different proteins can be seen in human plasma. How many biomarkers do you want? Math warning: more equations than figures. Archakov, A. et al., Proteomics 2009, 9, 1326‐1343. Unexplored territory: a catfish pathogen's proteome As genomic and proteomic tools become more powerful and cheaper per base or peptide, we can expect to see more papers like this one by Dumpala et al., focused on an organism of modest economic value. Each paper will, however, contribute a new niche with alternative adaptations for survival. In this case, we are introduced to Edwardsiella ictaluri, a Gram negative pathogen of farm‐raised channel catfish. Enteric septicemia of catfish is the most frequent disease of the commercially farmed catfish and appears in acute and chronic forms. For the work reported here, the bacteria were grown in culture, washed, lysed and separated by 2‐DE TOF/TOF or 2‐D LC‐MS/MS for peptide identification. The combined methods identified 788 unique proteins, including 73 ribosomal proteins, several protein synthesis factors, tRNA synthases and a number of other proteins that could be assigned by orthology to Escherichia coli or Edwardsiella tarda. Dumpala, P. R. et al., Proteomics 2009, 9, 1353‐1363.     

In this issue: Proteomics 5/2009

In this issue of Proteomics you will find the following highlighted articles: Heart (pump) broken? Hearts are pumps within pumps within channels and pumps. Calcium is pumped, potassium, sodium, amino acids, and electrons are all pumped, channeled or driven until, finally, blood is pumped. Failure of one or more pumps leads to a heart attack. This report from Zlatkovic et al. looks at the sub‐proteome associated with hypertensive failure of the K⁺ATP channel and associated cardiomyopathy that develops in KIR6.2 knock‐out mice. Out of >900 reproducible 2‐DE spots, 81 displayed significant over‐ or under‐expression, a number of which validated previously proposed interactions with the Kir6.2 channel. Two‐thirds were down‐regulations, including creatine kinase, adenylate kinase, and lactate dehydrogenase. A total of 114 proteins were ontologically mapped into the K⁺ATP‐dependent sub‐proteome and a role in hypertensive heart failure. Interaction mapping found >240 nodes and >1200 interactions/edges. A good foundation for future work. Zlatkovic, J. et al., Proteomics 2009, 9, 1314‐1325. The deeper you dig, the more you find A classical biochemist interested in protein‐protein interactions purifies his protein away from other proteins, seeking the highest “‐fold purification”. A proteomicist, on the other hand, looks for “consistent contamination” – i.e. association – of the protein of interest with other proteins. This requires high resolution separations and high accuracy concentration determinations. You can only work with species with concentrations above the detection limit (DL) for the detection method. 2‐DE MS has a DL of approximately 10^?8 M, LC‐MS/MS is ～10^?10 M and saturating Cy5 dye method is ～10^?13 M. Archakov et al. report on an atomic force microscope technique that can yield a DL of 10^?16 M when the target is irreversibly fixed to the bait to avoid the losses due to dissociation kinetics. At that level, over 1 000 000 different proteins can be seen in human plasma. How many biomarkers do you want? Math warning: more equations than figures. Archakov, A. et al., Proteomics 2009, 9, 1326‐1343. Unexplored territory: a catfish pathogen's proteome As genomic and proteomic tools become more powerful and cheaper per base or peptide, we can expect to see more papers like this one by Dumpala et al., focused on an organism of modest economic value. Each paper will, however, contribute a new niche with alternative adaptations for survival. In this case, we are introduced to Edwardsiella ictaluri, a Gram negative pathogen of farm‐raised channel catfish. Enteric septicemia of catfish is the most frequent disease of the commercially farmed catfish and appears in acute and chronic forms. For the work reported here, the bacteria were grown in culture, washed, lysed and separated by 2‐DE TOF/TOF or 2‐D LC‐MS/MS for peptide identification. The combined methods identified 788 unique proteins, including 73 ribosomal proteins, several protein synthesis factors, tRNA synthases and a number of other proteins that could be assigned by orthology to Escherichia coli or Edwardsiella tarda. Dumpala, P. R. et al., Proteomics 2009, 9, 1353‐1363.     

Exploring membrane and cytoplasm proteomic responses of Alkalimonas amylolytica N10 to different external pHs with combination strategy of de novo peptide sequencing

Identification of differentially proteomic responses to external pHs would pave an access for understanding of survival mechanisms of bacteria living at extreme pH environment. We cultured Alkalimonas amylolytica N10 (N10), a novel alkaliphilic bacterium found in Lake Chahannor, in media with three different pHs and extracted the correspondent membrane and cytoplasm proteins for proteomic analysis through 2‐DE. The differential 2‐DE spots corresponding to the altered pHs were delivered to MALDI TOF/TOF MS for protein identification. Since the genomic data of strain N10 was unavailable, we encountered a problem at low rate of protein identification with 18.1%. We employed, therefore, a combined strategy of de novo sequencing to analyze MS/MS signals generated from MALDI TOF/TOF MS. A significantly improved rate of protein identification was thus achieved at over than 70.0%. Furthermore, we extensively investigated the expression of these pH‐dependent N10 genes using Western blot and real‐time PCR. The conclusions drawn from immunoblot and mRNA measurements were mostly in agreement with the proteomic observations. We conducted the bioinformatic analysis to all the pH‐dependent N10 proteins and found that some membrane proteins participated in iron transport were differentially expressed as external pH elevated and most of differential proteins with increased or bell‐shape mode of pH‐dependence were involved in bioenergetic process and metabolism of carbohydrates, fatty acid, amino acids, and nucleotides. Our data thus provide a functional profile of the pH‐responsive proteins in alkaliphiles, leading to elucidation of alkaliphilic‐adaptive mechanism.     

Evaluation of sample preparation methods for the analysis of papaya leaf proteins through two‐dimensional gel electrophoresis

Introduction – A variety of sample preparation protocols for plant proteomic analysis using two‐dimensional gel electrophoresis (2‐DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. Objective – This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2‐DE. Methodology – Four sample preparation methods were tested: (1) phenol extraction and methanol–ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid–acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS–PAGE (1‐DE) and 2‐DE. Fifteen selected protein spots were trypsinised and analysed by matrix‐assisted laser desorption/ionisation time‐of‐flight tandem mass spectrometry (MALDI‐TOF‐MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. Results – Methods number 3 and 4 resulted in large quantities of protein with good 1‐DE separation and were chosen for 2‐DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. Conclusion – The described sample preparation method allows the proteomic analysis of papaya leaves by 2‐DE and mass spectrometry (MALDI‐TOF‐MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species. Copyright © 2009 John Wiley & Sons, Ltd.     

Biospecific irreversible fishing coupled with atomic force microscopy for detection of extremely low‐abundant proteins

In the absence of an analog of PCR for proteins, the concentration detection limit (DL) becomes a real challenge. The problem may be solved by means of a combination of biospecific irreversible fishing with atomic force microscopy (AFM). AFM offers the ability to register individual molecules and their complexes, while biospecific fishing takes advantage of an affine interaction between analyte molecules spread over a large volume of biomaterial and ligand molecules immobilized on the chip surface. Fishing may be conducted in Kd‐dependent reversible mode and in Kd‐independent irreversible mode. In this study, the DLs of two previously applied proteomic approaches were determined and compared to the DL of a newly developed analytical method. The first approach, based on MS analysis of biomaterial after 2‐DE or LC separation of proteins, attained a DL at the level of 10^?8–10^?10 M. The second approach, based on the optical biosensor analysis of molecular interactions in the format of proteomic microarrays, had a DL of 10^?9–10^?10 M. Our proposed method which combines biospecific fishing with AFM allowed us to attain DL values of 10^?11 M under reversible binding conditions and 10^?16 M under irreversible binding conditions.     

A proteomic approach for identifying cellular proteins interacting with erythropoietin in recombinant Chinese hamster ovary cells

Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull‐down assay was performed with dual‐tagged (N‐terminal GST‐ and C‐terminal hexahistidine‐tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual‐tagged EPO were then resolved by two‐dimensional gel electrophoresis (2DE) and identified by MALDI‐TOF MS/MS. A total of 27 protein spots including glucose‐regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull‐down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Proteomics analysis in Caenorhabditis elegans to elucidate the response induced by tyrosol,an olive phenol that stimulates longevity and stress resistance

Tyrosol (TYR, 2‐(4‐hydroxyphenyl)ethanol), one of the main phenols in olive oil and olive fruit, significantly strengthens resistance to thermal and oxidative stress in the nematode Caenorhabditis elegans and extends its lifespan. To elucidate the cellular functions regulated by TYR, we have used a proteomic procedure based on 2DE coupled with MS with the aim to identify the proteins differentially expressed in nematodes grown in a medium containing 250 μM TYR. After the comparison of the protein profiles from 250 μM TYR and from control, 28 protein spots were found to be altered in abundance (≥twofold). Analysis by MALDI‐TOF/TOF and PMF allowed the unambiguous identification of 17 spots, corresponding to 13 different proteins. These proteins were as follows: vitellogenin‐5, vitellogenin‐2, bifunctional glyoxylate cycle protein, acyl CoA dehydrogenase‐3, alcohol dehydrogenase 1, adenosylhomocysteinase, elongation factor 2, GTP‐binding nuclear protein ran‐1, HSP‐4, protein ENPL‐1 isoform b, vacuolar H ATPase 12, vacuolar H ATPase 13, GST 4. Western‐blot analysis of yolk protein 170, ras‐related nuclear protein, elongation factor 2, and vacuolar H ATPase H subunit supported the proteome evidence.     

A comprehensive proteome map of bovine cerebrospinal fluid

Cerebrospinal fluid (CSF) is considered as the most promising body fluid target for the discovery of biomarkers for early diagnosis of neurodegenerative diseases such as Creutzfeldt–Jakob disease in humans and bovine spongiform encephalopathy in cattle. For the recognition of disease‐associated changes in bovine CSF protein patterns, a detailed knowledge of this proteome is a prerequisite. The absence of a high‐resolution CSF proteome map prompted us to determine all bovine CSF protein spots that can be visualised on 2‐D protein gels. Using state‐of‐the‐art 2‐DE technology for proteome mapping of bovine ante mortem CSF combined with sensitive fluorescent protein staining and MALDI‐TOF/TOF MS for protein identification, a highly detailed 2‐DE map of the bovine CSF proteome was established. Besides the proteins mapped by earlier studies, this map contains 66 different proteins, including 58 which were not annotated in bovine 2‐DE CSF maps before.     

Concurrent Proteomic Fingerprinting and Molecular Analysis of Cyathostomins

Rapid, cost‐effective, efficient, and reliable helminth species identification is of considerable importance to understand host–parasite interactions, clinical disease, and drug resistance. Cyathostomins (Nematoda: Strongylidae) are considered to be the most important equine parasites, yet research on this group is hampered by the large number of 50 morphologically differentiated species, their occurrence in mixed infections with often more than 10 species and the difficulties associated with conventional identification methods. Here, MALDI‐TOF MS, previously successfully applied to identify numerous organisms, is evaluated and compared with conventional and molecular genetic approaches. A simple and robust protocol for protein extraction and subsequent DNA isolation allowing molecular confirmation of proteomic findings is developed, showing that MALDI‐TOF MS can discriminate adult stages of the two closely related cyathostomin species Cylicostephanus longibursatus and Cylicostephanus minutus. Intraspecific variability of proteomic profiles within morphospecies demonstrated an identification of morphospecies with an accuracy of close to 100%. In contrast, three genospecies within C. minutus and sex‐specific profiles within both morphospecies could not be reliably discriminated using MALDI‐TOF MS. In conclusion, MALDI‐TOF MS complemented by the molecular protocol is a reliable and efficient approach for cyathostomin species identification.     

Analysis of Human Plasma Proteome by 2DE‐ and 2D nanoLC‐Based Mass Spectrometry

Abstract

We compared the 2DE coupled to MALDI‐TOF‐MS and ESI‐MS/MS analysis (2DE‐MS) and the on‐line 2D nanoLC, followed by nanoESI‐MS/MS analysis (2DLC‐MS), for the separation and identification of proteins in high abundance protein‐depleted human plasma. Identification of proteins in the plasma by the two methods demonstrated that the majority of the identified protein set was unique to each method. Therefore, if a comprehensive coverage of the proteome identification is desired, it is ideal to apply both methods. The 2DE‐MS method is amenable to protein spot‐based quantitation, whereas the 2DLC‐MS method may provide an advantage of the high throughput application.     

Quantitative proteomic profiling reveals photosynthesis responsible for inoculum size dependent variation in Chlorella sorokiniana

High density cultivation is essential to industrial production of biodiesel from microalgae, which involves in variations of micro‐environment around individual cells, including light intensity, nutrition distribution, other abiotic stress and so on. To figure out the main limit factor in high inoculum cultivation, a quantitative proteomic analysis (iTRAQ‐on‐line 2‐D nano‐LC/MS) in a non‐model green microalga, Chlorella sorokiniana, under different inoculum sizes was conducted. The resulting high‐quality proteomic dataset consisted of 695 proteins. Using a cutoff of P < 0.05, 241 unique proteins with differential expression levels were identified between control and different inoculum sizes. Functional analysis showed that proteins participating in photosynthesis (light reaction) and Calvin cycle (carbon reaction pathway) had highest expression levels under inoculum size of 1 × 10⁶ cells mL^?1, and lowest levels under 1 × 10⁷ cells mL^?1. Canonical correlation analysis of the photosynthesis related proteins and metabolites biomarkers showed that a good correlation existed between them (canonical coefficient was 0.987), suggesting photosynthesis process greatly affected microalgae biodiesel productivity and quality. Proteomic study of C. sorokiniana under different illuminations was also conducted to confirm light intensity as a potential limit factor of high inoculum size. Nearly two thirds of proteins showed up‐regulation under the illumination of 70–110 µmol m^?2 s^?1, compared to those of 40 µmol m^?2 s^?1. This result suggested that by elegantly adjusting light conditions, high cell density cultivation and high biodiesel production might be achieved. Biotechnol. Bioeng. 2013; 110: 773–784. © 2012 Wiley Periodicals, Inc.     

Proteomics analysis of liver samples from puffer fish Takifugu rubripes exposed to excessive fluoride: An insight into molecular response to fluorosis

Comparative proteomics was performed to identify proteins in the liver of Takifugu rubripes in response to excessive fluoride exposure. Sixteen fish were randomly divided into a control group and an experimental group. The control group was raised in soft water alone (F^? = 0.4 mg/L), and the experimental group was raised in the same water with sodium fluoride at a high concentration of 35 mg/L. After 3 days, proteins were extracted from the fish livers and then subjected to two‐dimensional polyacrylamide gel electrophoresis analysis. The matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was applied to identify the proteins that were differentially expressed from the two groups of fish. Among an average of 816 and 918 proteins detected in the control and treated groups, respectively, 16 proteins were upregulated and 35 were downregulated (P < 0.01) in the fluoride‐treated group as compared with those in the control group. Twenty‐four highly differentially expressed proteins were further analyzed by MALDI‐TOF/TOF‐MS, and eight were identified by Mascot. These eight proteins include disulfide isomerase ER‐60, 4SNc‐Tudor domain protein, SMC3 protein, Cyclin D1, and mitogen‐activated protein kinase 10, as well as three unknown proteins. Consistent with their previously known functions, these identified proteins seem to be involved in apoptosis and other functions associated with fluorosis. These results will greatly contribute to our understanding of the effects of fluoride exposure on the physiological and biochemical functions of Takifugu and the toxicological mechanism of fluoride causing fluorosis in both fish and human. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:21–28, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20308     

Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild‐type and NOD mice

Non‐obese diabetic (NOD) mice exhibit impaired fertility and decreased litter size when compared to wild type (WT) mice. However, it is unclear why allogeneic pregnant NOD mice are prone to spontaneous embryo loss. Herein, two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry (MS) were used to detect differentially expressed proteins in the uterine lymphocytes isolated from these mice and WT BALB/c controls. We found 24 differentially expressed proteins. The differential expression of 10 of these proteins was further confirmed by Western blot analysis. Out of the 24 identified proteins, 20 were expressed in uterine lymphocytes of WT mice at a level at least 2 times higher than in NOD mice, whereas 4 were down‐regulated. Western blot analysis confirmed that 8 proteins were up‐regulated and 2 proteins were down‐regulated in WT mice compared with NOD mice, consistent with the results of 2‐DE and MS. Additionally, most of the highly expressed proteins in WT uterine lymphocytes were expressed at a significantly lower level in the corresponding splenic group (17/20). These results suggest that up‐regulated expression of these proteins may be specific to uterine lymphocytes. Reported functions of the highly expressed proteins affect key functions during pregnancy, including cell movement, cell cycle control, and metabolisms. Finally, we analyzed the constitutional ratio of CD3⁺ and CD49b⁺ cells in the isolated lymphocytes by flow cytometry. Our results suggest that the differentially expressed proteins may participate in the modulation of embryo implantation and early‐stage development of embryos, and subsequently influence pregnancy outcome. J. Cell. Biochem. 108: 447–457, 2009. © 2009 Wiley‐Liss, Inc.     

Proteomics of rice and Cochliobolus miyabeanus fungal interaction: Insight into proteins at intracellular and extracellular spaces

Necrotrophic fungal pathogen Cochliobolus miyabeanus causes brown spot disease in rice leaves upon infection, resulting in critical rice yield loss. To better understand the rice–C. miyabeanus interaction, we employed proteomic approaches to establish differential proteomes of total and secreted proteins from the inoculated leaves. The 2DE approach after PEG‐fractionation of total proteins coupled with MS (MALDI‐TOF/TOF and nESI‐LC‐MS/MS) analyses led to identification of 49 unique proteins out of 63 differential spots. SDS‐PAGE in combination with nESI‐LC‐MS/MS shotgun approach was applied to identify secreted proteins in the leaf apoplast upon infection and resulted in cataloging of 501 unique proteins, of which 470 and 31 proteins were secreted from rice and C. miyabeanus, respectively. Proteins mapped onto metabolic pathways implied their reprogramming upon infection. The enzymes involved in Calvin cycle and glycolysis decreased in their protein abundance, whereas enzymes in the TCA cycle, amino acids, and ethylene biosynthesis increased. Differential proteomes also generated distribution of identified proteins in the intracellular and extracellular spaces, providing a better insight into defense responses of proteins in rice against C. miyabeanus. Established proteome of the rice–C. miyabeanus interaction serves not only as a good resource for the scientific community but also highlights its significance from biological aspects.     

Assessment of oxidative stress biomarkers – neuroprostanes and dihomo-isoprostanes – in the urine of elite triathletes after two weeks of moderate-altitude training

This randomized and controlled trial investigated whether the increase in elite training at different altitudes altered the oxidative stress biomarkers of the nervous system. This is the first study to investigate four F₄-neuroprostanes (F₄-NeuroPs) and four F₂-dihomo-isoprostanes (F₂-dihomo-IsoPs) quantified in 24-h urine. The quantification was carried out by ultra high pressure liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-QqQ-MS/MS). Sixteen elite triathletes agreed to participate in the project. They were randomized in two groups, a group submitted to altitude training (AT, n?=?8) and a group submitted to sea level training (SLT) (n?=?8), with a control group (Cg) of non-athletes (n?=?8). After the experimental period, the AT group triathletes gave significant data: 17-epi-17-F_2t-dihomo-IsoP (from 5.2?±?1.4?μg/mL 24?h^?1 to 6.6?±?0.6?μg/mL 24?h^?1), ent-7(RS)-7-F_2t-dihomo-IsoP (from 6.6?±?1.7?μg/mL 24?h^?1 to 8.6?±?0.9?μg/mL 24?h^?1), and ent-7-epi-7-F_2t-dihomo-IsoP (from 8.4?±?2.2?μg/mL 24?h^?1 to 11.3?±?1.8?μg/mL 24?h^?1) increased, while, of the neuronal degeneration-related compounds, only 10-epi-10-F_4t-NeuroP (8.4?±?1.7?μg/mL 24?h^?1) and 10-F_4t-NeuroP (5.2?±?2.9?μg/mL 24?h^?1) were detected in this group. For the Cg and SLT groups, no significant changes had occurred at the end of the two-week experimental period. Therefore, and as the main conclusion, the training at moderate altitude increased the F₄-NeuroPs- and F₂-dihomo-isoPs-related oxidative damage of the central nervous system compared to similar training at sea level.