Cover Picture: Proteomics 2/2008

In this issue of Proteomics you will find the following highlighted articles: Particular particles pick out phosphopeptides promptly Phosphorylation/dephosphorylation is the most commonly used post‐translational signal in eukaryotic organisms. With a single site it might turn a pathway on or off, up or down; multiple site series can incrementally change the level of expression, effects can be direct or indirectly induced. Needless to say, phosphoproteins are extremely important subjects of study. Li et al. have developed a method for rapid collection and analysis of phosphopeptides: gallium oxide‐coated magnetic beads. Effective at very low phosphopeptide concentrations, a MALDI sample can be bound to the beads in 30 seconds. After a few washes, a small amount of the bead slurry is spotted on a MALDI plate, 2,5‐dihydroxybenzoic acid spotted as matrix, then, “Fire away!” The gallium oxide beads dramatically out perform silica‐Fe beads, Fe+3‐IMAC resin, and TiO2 beads. Li, Y. et al., Proteomics 2008, 8, 238–249. Plasma butyrylcholinesterase: multiple N‐glycan sites support multiple roles Cholinesterases have been of interest since their discovery in the early 1930’s. Acetylcholinesterase is the target of insecticides and nerve gases. Butyrylcholinesterase (BChE) plays a variety of roles because of its “relaxed” substrate requirements, able to detoxify heroin and aspirin as well as choline‐containing molecules. Of particular interest is its function as a scavenger of organophosphates to protect nerve‐associated acetylcholinesterase. Kolarich et al. explored the complexities of glycosylation of BChE, with 9 of 10 potential N‐glycosylation sites occupied in the 85 kDa protein. Of the variety of techniques applied to elucidating the glycan structures at particular sites, perhaps the most informative was porous graphitic carbon LC/MS. It yielded more information in 80 minutes than an overnight “classical” procedure. No evidence of O‐glycosylation or other post‐translational modifications were seen. Kolarich, D. et al., Proteomics 2008, 8, 254–263. DIGE digs up skeletal muscle fate The gradual loss of strength and muscle mass is a normal event in aging, measurable by published statistics for professional athletes, or by how long we take to push away from the table. Sarcopenia is the drastic form of muscle loss, resulting in severe impairment. Doran et al. selected the rat model of muscle mass loss between 3 months (young adult) and 30 months (old) to study, avoiding confounding human variables. Applying DIGE/MALDI‐TOF to gastrocnemius samples, they found 2493 spots, of which 69 exhibited dramatic up‐ or down‐regulation. The functional changes suggested by the quantitative changes included increased dependence on aerobic oxidative metabolism and fibre remodeling, probably due to impaired fibre repair. These findings were confirmed by Western blots and fluorescent confocal microscopy and concur with other published studies. Doran, P. et al., Proteomics 2008, 8, 364–377.     

In this issue: Proteomics 2/2008

In this issue of Proteomics you will find the following highlighted articles: Particular particles pick out phosphopeptides promptly Phosphorylation/dephosphorylation is the most commonly used post‐translational signal in eukaryotic organisms. With a single site it might turn a pathway on or off, up or down; multiple site series can incrementally change the level of expression, effects can be direct or indirectly induced. Needless to say, phosphoproteins are extremely important subjects of study. Li et al. have developed a method for rapid collection and analysis of phosphopeptides: gallium oxide‐coated magnetic beads. Effective at very low phosphopeptide concentrations, a MALDI sample can be bound to the beads in 30 seconds. After a few washes, a small amount of the bead slurry is spotted on a MALDI plate, 2,5‐dihydroxybenzoic acid spotted as matrix, then, “Fire away!” The gallium oxide beads dramatically out perform silica‐Fe beads, Fe+3‐IMAC resin, and TiO2 beads. Li, Y. et al., Proteomics 2008, 8, 238–249. Plasma butyrylcholinesterase: multiple N‐glycan sites support multiple roles Cholinesterases have been of interest since their discovery in the early 1930’s. Acetylcholinesterase is the target of insecticides and nerve gases. Butyrylcholinesterase (BChE) plays a variety of roles because of its “relaxed” substrate requirements, able to detoxify heroin and aspirin as well as choline‐containing molecules. Of particular interest is its function as a scavenger of organophosphates to protect nerve‐associated acetylcholinesterase. Kolarich et al. explored the complexities of glycosylation of BChE, with 9 of 10 potential N‐glycosylation sites occupied in the 85 kDa protein. Of the variety of techniques applied to elucidating the glycan structures at particular sites, perhaps the most informative was porous graphitic carbon LC/MS. It yielded more information in 80 minutes than an overnight “classical” procedure. No evidence of O‐glycosylation or other post‐translational modifications were seen. Kolarich, D. et al., Proteomics 2008, 8, 254–263. DIGE digs up skeletal muscle fate The gradual loss of strength and muscle mass is a normal event in aging, measurable by published statistics for professional athletes, or by how long we take to push away from the table. Sarcopenia is the drastic form of muscle loss, resulting in severe impairment. Doran et al. selected the rat model of muscle mass loss between 3 months (young adult) and 30 months (old) to study, avoiding confounding human variables. Applying DIGE/MALDI‐TOF to gastrocnemius samples, they found 2493 spots, of which 69 exhibited dramatic up‐ or down‐regulation. The functional changes suggested by the quantitative changes included increased dependence on aerobic oxidative metabolism and fibre remodeling, probably due to impaired fibre repair. These findings were confirmed by Western blots and fluorescent confocal microscopy and concur with other published studies. Doran, P. et al., Proteomics 2008, 8, 364–377.     

Proteomic analysis of β‐catenin activation in mouse liver by DIGE analysis identifies glucose metabolism as a new target of the Wnt pathway

The Wnt/β‐catenin signaling pathway has been increasingly implicated in liver development and physiology. Aberrant activation of this pathway is one of the major genetic events observed during the process of human HCC development. To gain insight into the mechanism underlying β‐catenin action in the liver, we conducted a quantitative differential proteomic analysis using 2‐D DIGE combined with MS, in mice with liver‐specific deletion of Apc resulting in acute activation of β‐catenin signaling (Apc^KOliv mice). We identified 94 protein spots showing differential expression between mutant Apc^KOliv and control mice, corresponding to 56 individual proteins. Most of the proteins identified were associated with metabolic pathways, such as ammonia and glucose metabolism. Our analysis showed an increase in lactate dehydrogenase activity together with a downregulation of two mitochondrial ATPase subunits (ATP5a1 and ATP5b). These observations indicate that β‐catenin signaling may induce a shift in the glucose metabolism from oxidative phosphorylation to glycolysis, known as the “Warburg effect”. Imaging with ¹⁸F‐fluoro‐2‐deoxy‐D ‐glucose‐positron emission tomography suggests that the specific metabolic reprogramming induced by β‐catenin in the liver does not imply the first step of glycolysis. This observation may explain why some HCCs are difficult to assess by fluoro‐2‐deoxy‐D ‐glucose‐positron emission tomography imaging.     

Phosphoproteomic analysis of wild‐type and antimony‐resistant Leishmania braziliensis lines by 2D‐DIGE technology

Protein phosphorylation is one of the most studied post‐translational modifications that is involved in different cellular events in Leishmania. In this study, we performed a comparative phosphoproteomics analysis of potassium antimonyl tartrate (SbIII)‐resistant and ‐susceptible lines of Leishmania braziliensis using a 2D‐DIGE approach followed by MS. In order to investigate the differential phosphoprotein abundance associated with the drug‐induced stress response and SbIII‐resistance mechanisms, we compared nontreated and SbIII‐treated samples of each line. Pair wise comparisons revealed a total of 116 spots that showed a statistically significant difference in phosphoprotein abundance, including 11 and 34 spots specifically correlated with drug treatment and resistance, respectively. We identified 48 different proteins distributed into seven biological process categories. The category “protein folding/chaperones and stress response” is mainly implicated in response to SbIII treatment, while the categories “antioxidant/detoxification,” “metabolic process,” “RNA/DNA processing,” and “protein biosynthesis” are modulated in the case of antimony resistance. Multiple sequence alignments were performed to validate the conservation of phosphorylated residues in nine proteins identified here. Western blot assays were carried out to validate the quantitative phosphoproteome analysis. The results revealed differential expression level of three phosphoproteins in the lines analyzed. This novel study allowed us to profile the L. braziliensis phosphoproteome, identifying several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite.     

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.     

Cover Picture: Proteomics 1/2008

In this issue of Proteomics you will find the following highlighted articles: Arachnophilia: A Charlotte working on the web In the children’s book Charlotte’s Web, a spider communicates with a pig by weaving messages into her web. In this Technical Brief, Mayer’s spider is the intermediate, a program taking queries about the protein world and weaving relevant information from the www’s libraries and databases into spreadsheets. PIC (Protein Information Crawler) can link directly to a number of databases including BLAST, SMART, PROSITE, and CDD. Selected data is deposited in an Excel spreadsheet or HTML table for sorting and browsing. The system is customizable to anyone with minimal programming skills in LabView G, an easy‐to‐learn graphical language. Using PIC reduced the initial data search for a system of ～1000 neural proteins from 8 wks to 2 days. The software is free. Mayer, U., Proteomics 2008, 8, 42–44. Hard heart, soft heart: analyzing tropomyosin links to types of cardiomyopathy I don’t know if the type of a heart patient’s cardiomyopathy has been diagnosed by behavioral observations but Warren et al. examined the behavior of tropomyosin on improved 2‐D PAGE and 2‐D DIGE separations. First dimension separations were run on 18‐cm long narrow range (pH 4.5 to pH 5.5) IPG strips. Second dimension gels were 16 cm wide, 1 mm thick, and 8 cm long. Ends of the IPG strips were trimmed off to fit the vertical gel. The equilibrated strip was put in place without agarose on top of stacking and resolving gels that included 10% glycerol and, in the stacking gel, 15% N,N’‐diallyltartardiamide to ensure efficient transfer of the protein from the first‐ to the second‐dimension gel. With these changes they were able to distinguish wild type tropomyosin from an E54K mutant and phosphorylated from unphosphorylated tropomyosin, potentially key prognostic clues. Warren, C. M. et al., Proteomics 2008, 8, 100–105. Moo‐ving into ART: Cows lead the way Cow ART is not the product of a bovine Moonet or Moodigliani, it is “Assist­ed Reproductive Technology.” Not simply artificial insemination, ART includes somatic cell nuclear transfer and other advanced techniques which are critical to creating breeding herds with “elite” genetics. But the success rate is not what was expected or required for effective use. Riding et al. apply proteome analysis techniques to establish a foundation for pregnancy progress biomarkers. Ruminants have two fluid‐filled sacs, amniotic and allantoic, that are critical to fetal development. After developing an improved sample prep procedure, the 5–50 kDa fraction of the allantoic proteome was analyzed. Some 139 proteins were identified and ontologically classified into nine functional groups. Too little amniotic fluid was recovered for thorough analysis but the two fluids were clearly distinguishable at 45 days post‐conception. Riding, G. et al., Proteomics 2008, 8, 160–177.     

In this issue: Proteomics 1/2008

In this issue of Proteomics you will find the following highlighted articles: Arachnophilia: A Charlotte working on the web In the children’s book Charlotte’s Web, a spider communicates with a pig by weaving messages into her web. In this Technical Brief, Mayer’s spider is the intermediate, a program taking queries about the protein world and weaving relevant information from the www’s libraries and databases into spreadsheets. PIC (Protein Information Crawler) can link directly to a number of databases including BLAST, SMART, PROSITE, and CDD. Selected data is deposited in an Excel spreadsheet or HTML table for sorting and browsing. The system is customizable to anyone with minimal programming skills in LabView G, an easy‐to‐learn graphical language. Using PIC reduced the initial data search for a system of ～1000 neural proteins from 8 wks to 2 days. The software is free. Mayer, U., Proteomics 2008, 8, 42–44. Hard heart, soft heart: analyzing tropomyosin links to types of cardiomyopathy I don’t know if the type of a heart patient’s cardiomyopathy has been diagnosed by behavioral observations but Warren et al. examined the behavior of tropomyosin on improved 2‐D PAGE and 2‐D DIGE separations. First dimension separations were run on 18‐cm long narrow range (pH 4.5 to pH 5.5) IPG strips. Second dimension gels were 16 cm wide, 1 mm thick, and 8 cm long. Ends of the IPG strips were trimmed off to fit the vertical gel. The equilibrated strip was put in place without agarose on top of stacking and resolving gels that included 10% glycerol and, in the stacking gel, 15% N,N’‐diallyltartardiamide to ensure efficient transfer of the protein from the first‐ to the second‐dimension gel. With these changes they were able to distinguish wild type tropomyosin from an E54K mutant and phosphorylated from unphosphorylated tropomyosin, potentially key prognostic clues. Warren, C. M. et al., Proteomics 2008, 8, 100–105. Moo‐ving into ART: Cows lead the way Cow ART is not the product of a bovine Moonet or Moodigliani, it is “Assist­ed Reproductive Technology.” Not simply artificial insemination, ART includes somatic cell nuclear transfer and other advanced techniques which are critical to creating breeding herds with “elite” genetics. But the success rate is not what was expected or required for effective use. Riding et al. apply proteome analysis techniques to establish a foundation for pregnancy progress biomarkers. Ruminants have two fluid‐filled sacs, amniotic and allantoic, that are critical to fetal development. After developing an improved sample prep procedure, the 5–50 kDa fraction of the allantoic proteome was analyzed. Some 139 proteins were identified and ontologically classified into nine functional groups. Too little amniotic fluid was recovered for thorough analysis but the two fluids were clearly distinguishable at 45 days post‐conception. Riding, G. et al., Proteomics 2008, 8, 160–177.     

Highly sensitive saturation labeling reveals changes in abundance of cell cycle‐associated proteins and redox enzyme variants during oocyte maturation in vitro

Oocyte maturation is a complex process and a critical issue in assisted reproduction techniques (ART) in humans and other mammals. We used a sensitive 2‐D DIGE saturation labeling approach including an internal pooled standard for quantitative proteome profiling of immature versus in vitro matured bovine oocytes in six independent samples. The study comprised 48 2D gel images representing 24 DIGE experiments. From 250 ng sample analyzed per gel, quantitative analysis revealed an average of 2244 spots in pH 4–7 images and 1291 spots in pH 6–9 images. Thirty‐eight spots with different intensities were detected in total. Spots of a preparative gel from 2200 oocytes were identified by nano‐LC‐MS/MS analysis. The ten spots which could be unambiguously identified include the Ca²⁺‐binding protein translationally controlled tumor protein, enzymes of the Krebs and pentose phosphate cycles, clusterin, 14‐3‐3 ?, elongation factor‐1 gamma, and redox enzymes such as polymorphic forms of GST Mu 5 and peroxiredoxin‐3. The cellular distribution of two proteins was determined by confocal laser scanning microscopy. The interesting protein candidates identified by this study may help to improve the in vitro maturation process in order to increase the rate of successful in vitro fertilization and other ART in cattle and other mammals.     

Physiological and proteome study of sunflowers exposed to a polymetallic constraint

The new energy requirements of the growing world population together with the actual ecological trend of phytoremediation have made challenging the cultivation of energetic crops on nonagricultural lands, such as those contaminated with trace elements. In this study, phenotypical characterization and biochemical analyses were combined to emphasize the global response of young sunflowers (Helianthus annuus L.) grown in hydroponic media contaminated with different Cd, Ni, and Zn concentrations. Leaves and roots of sunflowers reaching the stage “2‐extended leaves” and exposed to different trace metal concentrations were harvested and analyzed by 2D‐DIGE in order to study in depth the molecular responses of the young plants upon the polymetallic exposure. Proteomics confirmed the observed global reduction in growth and development. If photosynthetic light reactions and carbon metabolism were the most affected in leaves, in roots significant disruptions were observed in proteins involved in respiration, oxidative balance, protein and gene expression, and in the induction of programmed cell death. Elemental analyses of the plantlets indicated a profound impact of the treatment resulting in misbalance in essential micronutrients. Altogether, this study highlights the sensitivity of the sunflower to a polymetallic pollution and indicates that its use as a remediative tool of trace element polluted soils is limited.     

Cover Picture: Proteomics 6/2009

In this issue of Proteomics you will find the following highlighted articles: Keeping up with the lung cancers You're in good company if you smoke and develop lung cancer. The World Health Organization estimates 1.2 million new cases occur every year. On the other hand, 1.1 million people die from it every year‐bummer. One reason for the high death rate is the frequent development of resistance to several of the most commonly used drugs simultaneously. Multiple drug resistance (MDR) is the major cause of chemotherapeutic failure. Keenan et al. explored the proteomic changes associated with MDR failure (adriamycin) in a cultured lung cancer cell line (DLKP) and several subtypes. Adriamycin normally kills by blocking replication at DNA gyrase and by generating reactive oxygen species that lead to apoptosis. Proteomes were examined by 2‐D DIGE. Approximately 80 proteins displayed quantitative shifts, 32 showed a correlation with resistance, 24 being linked positively to resistance, 6 correlated negatively. Some known targets did not appear on the 2‐D maps consistently. Keenan, J. et al., Proteomics 2009, 9, 1556‐1566. An image of spit Spitting images have been around for a long time. The phrase is possibly human‐kind's first recognition of genetically transmitted traits. Proteomic analysis of saliva has only developed recently. The question raised by Walz et al. here is “What is the possible contribution of saliva to the high level of infection by Helicobacter pylori?” H. pylori is known to have extracellular adhesins that bind to a number of salivary proteins. A convenient way to detect targets of adhesins was found to be incubating 1‐D and 2‐D PAGE Western blots with an overlay of whole H. pylori. Targets detected included mucins, sialic acid‐containing glycoproteins, fucose‐containing blood group antigens and each pair of salivary glands had a different binding pattern. Walz, A. et al., Proteomics 2009, 9, 1582‐1592. Mix'em up, folks Conventional analytical chemical identifications frequently yield a characteristic spectrum of peaks for particular compounds on particular instruments. Just look up the observed spectrum in the “library” of standard spectra for identification. It is not so simple for proteins. Because of the size of a potential proteomic peptide library and the diversity of instruments used, most often the observed spectrum is compared to a theoretical spectrum for a peptide of interest. Ahrné et al. combine the two for improved performance. First they run the spectrum of interest through an exhaustive proteome search program (Phenyx), then through a sensitive library search (SpectraST) of the highest scoring sequences in the previous Phenyx search plus a number of controls. In the first (relatively simple) test, Phenyx matched 362 spectra, SpectraST made 639 matches at the same error detection level. In a more complex test, Phenyx generated >1000 hits, SpectraST 1304 hits. Ahrné, E. et al., Proteomics 2009, 9, 1731‐1736.     

In this issue: Proteomics 6/2009

In this issue of Proteomics you will find the following highlighted articles: Keeping up with the lung cancers You're in good company if you smoke and develop lung cancer. The World Health Organization estimates 1.2 million new cases occur every year. On the other hand, 1.1 million people die from it every year‐bummer. One reason for the high death rate is the frequent development of resistance to several of the most commonly used drugs simultaneously. Multiple drug resistance (MDR) is the major cause of chemotherapeutic failure. Keenan et al. explored the proteomic changes associated with MDR failure (adriamycin) in a cultured lung cancer cell line (DLKP) and several subtypes. Adriamycin normally kills by blocking replication at DNA gyrase and by generating reactive oxygen species that lead to apoptosis. Proteomes were examined by 2‐D DIGE. Approximately 80 proteins displayed quantitative shifts, 32 showed a correlation with resistance, 24 being linked positively to resistance, 6 correlated negatively. Some known targets did not appear on the 2‐D maps consistently. Keenan, J. et al., Proteomics 2009, 9, 1556‐1566. An image of spit Spitting images have been around for a long time. The phrase is possibly human‐kind's first recognition of genetically transmitted traits. Proteomic analysis of saliva has only developed recently. The question raised by Walz et al. here is “What is the possible contribution of saliva to the high level of infection by Helicobacter pylori?” H. pylori is known to have extracellular adhesins that bind to a number of salivary proteins. A convenient way to detect targets of adhesins was found to be incubating 1‐D and 2‐D PAGE Western blots with an overlay of whole H. pylori. Targets detected included mucins, sialic acid‐containing glycoproteins, fucose‐containing blood group antigens and each pair of salivary glands had a different binding pattern. Walz, A. et al., Proteomics 2009, 9, 1582‐1592. Mix'em up, folks Conventional analytical chemical identifications frequently yield a characteristic spectrum of peaks for particular compounds on particular instruments. Just look up the observed spectrum in the “library” of standard spectra for identification. It is not so simple for proteins. Because of the size of a potential proteomic peptide library and the diversity of instruments used, most often the observed spectrum is compared to a theoretical spectrum for a peptide of interest. Ahrné et al. combine the two for improved performance. First they run the spectrum of interest through an exhaustive proteome search program (Phenyx), then through a sensitive library search (SpectraST) of the highest scoring sequences in the previous Phenyx search plus a number of controls. In the first (relatively simple) test, Phenyx matched 362 spectra, SpectraST made 639 matches at the same error detection level. In a more complex test, Phenyx generated >1000 hits, SpectraST 1304 hits. Ahrné, E. et al., Proteomics 2009, 9, 1731‐1736.     

Retracted: A proteomic approach for investigation of photosynthetic apparatus in plants

Retraction: The following article from Proteomics, “A proteomic approach for investigation of photosynthetic apparatus in plants” by C. Ciambella, P. Roepstorff, E.M. Aro and L. Zolla, published online on 28 January 2005 in the Wiley Online Library ( http://onlinelibrary.wiley.com/doi/10.1002/pmic.200401129/full ), has been retracted by agreement between the authors, the Editor‐in‐Chief and Wiley‐VCH GmbH & Co. KGaA. The retraction has been agreed due to the similarity of Figure 4 in this article and an image from an article by B. Granvogl and L.A. Eichacker which was originally submitted to Proteomics on November 1^st, 2002 and which was finally published online on 6 June 2006 in the Wiley Online Library ( http://onlinelibrary.wiley.com/doi/10.1002/pmic.200500924/full ) as Figure 1 in Proteomics, “Mapping the proteome of thylakoid membranes by de novo sequencing of intermembrane peptide domains” by B. Granvogl, V. Reisinger and L.A. Eichacker.     

Helicobacter pylori outer inflammatory protein A (OipA) suppresses apoptosis of AGS gastric cells in vitro

Outer inflammatory protein A (OipA) is an important virulence factor associated with gastric cancer and ulcer development; however, the results have not been well established and turned out to be controversial. This study aims to elucidate the role of OipA in Helicobacter pylori infection using clinical strains harbouring oipA “on” and “off” motifs. Proteomics analysis was performed on AGS cell pre‐infection and postinfection with H. pylori oipA “on” and “off” strains, using liquid chromatography/mass spectrometry. AGS apoptosis and cell cycle assays were performed. Moreover, expression of vacuolating cytotoxin A (VacA) was screened using Western blotting. AGS proteins that have been suggested previously to play a role or associated with gastric disease were down‐regulated postinfection with oipA “off” strains comparing to oipA “on” strains. Furthermore, oipA “off” and ΔoipA cause higher level of AGS cells apoptosis and G0/G1 cell‐cycle arrest than oipA “on” strains. Interestingly, deletion of oipA increased bacterial VacA production. The capability of H. pylori to induce apoptosis and suppress expression of proteins having roles in human disease in the absence of oipA suggests that strains not expressing OipA may be less virulent or may even be protective against carcinogenesis compared those expressing OipA. This potentially explains the higher incidence of gastric cancer in East Asia where oipA “on” strains predominates.     

Proteome‐wide search for PP2A substrates in fission yeast

PP2A (protein phosphatase 2A) is a major phosphatase in eukaryotic cells that plays an essential role in many processes. PP2A mutations in Schizosaccharomyces pombe result in defects of cell cycle control, cytokinesis and morphogenesis. Which PP2A substrates are responsible for these changes is not known. In this work, we searched for PP2A substrates in S. pombe using two approaches, 2D‐DIGE analysis of PP2A complex mutants and identification of PP2A interacting proteins. In both cases, we used MS to identify proteins of interest. In the DIGE experiment, we compared proteomes of wild‐type S. pombe, deletion of pta2, the phosphoactivator of the PP2A catalytic subunit, and pab1–4, a mutant of B‐type PP2A regulatory subunit. A total of 1742 protein spots were reproducibly resolved by 2D‐DIGE and 51 spots demonstrated significant changes between PP2A mutants and the wild‐type control. MS analysis of these spots identified 27 proteins that include key regulators of glycerol synthesis, carbon metabolism, amino acid biosyntesis, vitamin production, and protein folding. Importantly, we independently identified a subset of these proteins as PP2A binding partners by affinity precipitation, suggesting they may be direct targets of PP2A. We have validated our approach by demonstrating that phosphorylation of Gpd1, a key enzyme in glycerol biogenesis, is regulated by PP2A and that ability of cells to respond to osmotic stress by synthesizing glycerol is compromised in the PP2A mutants. Our work contributes to a better understanding of PP2A function and identifies potential PP2A substrates.     

In this issue: Proteomics 5/2008

In this issue of Proteomics you will find the following highlighted articles: When is a stain not a stain? When it's dyeing! [Dumb proteomics joke!] This silly riddle is actually relat­ed to a recurrent question in proteomics: when is the best time to apply detection reagents to proteins for quantitative analysis? (a) pre‐electrophoresis labeling with DIGE/Cy‐type of covalent stains, or (b) post‐electrophoresis staining with silver, Sypro Ruby or Deep Purple? Karp et al. explore the question using a bacterial extract as a typical sample, DIGE Cy labels, and Deep Purple. It gets more complex when they have to deal with the “missingness” of spots: just because a spot doesn’t show up doesn’t mean it is not there, there just may not be enough to detect. Progenesis SameSpots software was used to analyze images for missing spots. In the end, DIGE gave better sensitivity as previously reported, and fewer missing spots. Deep Purple was more competitive when analyzed with SameSpots software. Karp, N. A. et al., Proteomics 2008, 8, 948–960. Your own best enemy? If there weren’t one maverick, black sheep, rebel, outlaw, eccentric, or rotten apple in most families, a lot of novels would never have been written. Mammalian immune systems seem to have the same structure – they mostly target enemies of the body but there always seem to be a few maverick antibodies that are targeted at their own body’s antigens. Servettaz et al. take up proteomic tools to identify the targets of the anti‐self antibodies expressed by apparently healthy individuals. Using umbilical cord endothelial cells as a source of antigens, the authors found 884 spots by ­2‐­DE, and 61 ± 25 of those were recognized by serum IgGs. All 12 sera tested recognized 11 antigens derived from 6 proteins. There were 3 cytoskeletal, 2 glycolytic, and 1 disulfide isomerase protein seen. These were confirmed by immunoblotting of 2‐D gels and identification by in‐gel tryptic digestion and MALDI‐TOF MS. Servettaz, A. et al., Proteomics 2008, 8, 1000–1008. Signature in scraps from kidney growth stages You can tell a lot about the quality of a new building, residential or commercial, by what doesn’t go into it. The scraps of lumber, pieces of masonry, lengths and varieties of cables are all revealing. Lee et al. watch the final maturation of the rat urinary tract by proteomic analysis of the debris found in urine over time. Taking special care not to mix adult and neonatal urine, they examined four samples over 2 weeks after birth and one at maturity, >30 d. Using nano‐ESI‐LC‐MS/MS technology, six proteins were found in all samples, 30 were adult specific. Proteins were further characterized by large format 1‐ and 2‐DE, immunoblots, and immunofluorescent analysis of tissue sections. Days 1, 3, and 7 had 37% of proteins in common whereas days 7, 14 and >30 shared only 7.4% of proteins. Levels of fibronectin and location of E‐cadherin expression shifted during maturation. Lee, R. S. et al., Proteomics 2008, 8, 1097–1112.     

Cover Picture: Proteomics 1/2009

In this issue of Proteomics you will find the following highlighted articles: How many tries before you get it right? British Prime Minister Benjamin Disraeli is reputed to have stated that “There are three types of lies: lies, damned lies and statistics.” As those immersed in bioinformatics have recognized, though they may be slippery characters, statistics are the only way some information can be extracted from an experimental structure. One of the recurring problems is the question of how many samples need to be tested to get a reasonable, reliable result. This is particularly important when samples are difficult to get, require arduous preparation, or yield only small amounts. These experiments are generally multidimensional. In this article Cairns et al., examine the number of mass spectrometry samples that are required for a quantitative answer in a biomarker search. They evaluate MALDI‐TOF and SELDI‐TOF data for sources and amounts of variability on a pilot scale (biological and technical particularly) which allows them to calculate the number of samples required for a valid full‐scale screen. Cairns, D. A. et al., Proteomics 2009, 9, 74‐86. Double‐barreled proteomic run on embryonic stem cell membranes Embryonic stem cells (ESC) appear to be as close to the fountain of youth as most of us can reasonably expect to get in this lifetime. How close they come to being a “silver bullet” for cancer and other diseases is yet to be determined. Intoh et al., have taken a major step forward in improving our understanding of ESC control and maintenance. They applied 2‐D DIGE and trypsin digestion + iTRAQ labeling to identify membrane and membrane‐associated proteins in mouse ESCs that had or had not been exposed to leukemia inhibitory factor, a factor which maintains pluripotency in ESCs. Some 338 membrane and membrane‐associated proteins, up‐ or down‐regulated, were identified and assigned to functional groups. Intoh, A. et al., Proteomics 2009, 9, 126‐137. H, M, L You see these three letters on a variety of simple controllers: pump speed, temperature, under‐desk foot warmers, etc. Now you can hope to see them soon on bottles in a cell mass isotope labeling kit. Schwanhäusser et al., describe here a protocol for following levels of protein expression in array volumes and numbers with array simplicity. They pulse label samples with Heavy, Medium, or Light amino acids. Pulse‐labeling has been used for determining protein turnover rates for eons but with a quantitation problem for translation: did the ratio change because the numerator changed or because the denominator changed? The answer comes from labeling the untreated control with the M amino acid, then mixing M+H or M+L samples before fractionating by SDS‐PAGE and high‐resolution LC‐MS/MS. It worked for cell fractions (HeLa) as well as whole cells (yeast). Schwanhäusser, B. et al., Proteomics 2009, 9, 205‐209.     

Cover Picture: Proteomics 5/2008

In this issue of Proteomics you will find the following highlighted articles: When is a stain not a stain? When it's dyeing! [Dumb proteomics joke!] This silly riddle is actually relat­ed to a recurrent question in proteomics: when is the best time to apply detection reagents to proteins for quantitative analysis? (a) pre‐electrophoresis labeling with DIGE/Cy‐type of covalent stains, or (b) post‐electrophoresis staining with silver, Sypro Ruby or Deep Purple? Karp et al. explore the question using a bacterial extract as a typical sample, DIGE Cy labels, and Deep Purple. It gets more complex when they have to deal with the “missingness” of spots: just because a spot doesn’t show up doesn’t mean it is not there, there just may not be enough to detect. Progenesis SameSpots software was used to analyze images for missing spots. In the end, DIGE gave better sensitivity as previously reported, and fewer missing spots. Deep Purple was more competitive when analyzed with SameSpots software. Karp, N. A. et al., Proteomics 2008, 8, 948–960. Your own best enemy? If there weren’t one maverick, black sheep, rebel, outlaw, eccentric, or rotten apple in most families, a lot of novels would never have been written. Mammalian immune systems seem to have the same structure – they mostly target enemies of the body but there always seem to be a few maverick antibodies that are targeted at their own body’s antigens. Servettaz et al. take up proteomic tools to identify the targets of the anti‐self antibodies expressed by apparently healthy individuals. Using umbilical cord endothelial cells as a source of antigens, the authors found 884 spots by ­2‐­DE, and 61 ± 25 of those were recognized by serum IgGs. All 12 sera tested recognized 11 antigens derived from 6 proteins. There were 3 cytoskeletal, 2 glycolytic, and 1 disulfide isomerase protein seen. These were confirmed by immunoblotting of 2‐D gels and identification by in‐gel tryptic digestion and MALDI‐TOF MS. Servettaz, A. et al., Proteomics 2008, 8, 1000–1008. Signature in scraps from kidney growth stages You can tell a lot about the quality of a new building, residential or commercial, by what doesn’t go into it. The scraps of lumber, pieces of masonry, lengths and varieties of cables are all revealing. Lee et al. watch the final maturation of the rat urinary tract by proteomic analysis of the debris found in urine over time. Taking special care not to mix adult and neonatal urine, they examined four samples over 2 weeks after birth and one at maturity, >30 d. Using nano‐ESI‐LC‐MS/MS technology, six proteins were found in all samples, 30 were adult specific. Proteins were further characterized by large format 1‐ and 2‐DE, immunoblots, and immunofluorescent analysis of tissue sections. Days 1, 3, and 7 had 37% of proteins in common whereas days 7, 14 and >30 shared only 7.4% of proteins. Levels of fibronectin and location of E‐cadherin expression shifted during maturation. Lee, R. S. et al., Proteomics 2008, 8, 1097–1112.