In this issue: Proteomics 17/2008

In this issue of Proteomics you will find the following highlighted articles: Slidin' and slipin': Substrates for autoantibody antigen arrays Proteins do not have a reputation for being well‐behaved. Given the number of sequence permutations possible for a particular length, it is no wonder that protein arrays have a notorious history. Balboni et al. report here on a systematic survey of supports and application methods for autoantibody antigen arrays. These arrays are central to studies of autoimmune diseases such as juvenile‐onset (type I) diabetes, rheumatoid arthritis, multiple sclerosis, etc. Over 20 commercial and home‐made slides were tested for background, smearing, streaking, adherence, and intra‐ and inter‐slide variability (CVs). FAST® slides were ranked the best on the CV scores. Also acceptable were PATH® and SuperEpoxy2 slides. The authors note that other slide types may be better for specific antigens or detection methods. Balboni, I. et al, Proteomics 2008, 8, 3443–3449. Cheesy target for high resolution proteomics We are what we eat and sometimes that includes the leftovers from other organisms. “Yecch!” you say, but these are the products of a fermentome (to coin a new name), those proteins and organisms that ferment our food – grapes to wine, milk to yogurt, etc. – processes that need to be well understood for food safety and quality. Soufi et al. explore the phosphoproteome of Lactococcus lactis, an important commercial strain of bacteria, used in making a variety of fermented food products. L. lactis exhibits site‐specific phosphorylation of serine, threonine and tryptophan residues similar to that found in eukaryotes. Unlike eukaryotes, bacteria usually have only one phosphorylation site per protein. The evidence presented suggests protein phosphorylation is a means to regulate gene expression in bacteria, albeit on a smaller scale than in higher organisms. Soufi, B. et al, Proteomics 2008, 8, 3486–3493. To see or not to see: That is not a question One of the most frequent healing complications of surgical repair of a detached retina is the overgrowth of membranes on both surfaces of the retina and the back side of the vitreous body (PVR) which can contract and rip the underlying tissue loose, creating major vision problems. Yu et al. applied proteomic tools to the problem and found the explanation lay in misregulation of a number of cytoskeleton and metabolism genes. Normal, moderate and severe PVR vitreous and serum samples were treated with trypsin and analyzed by strong cation exchange‐ and reverse phase‐chromatography then nanoelectrospray‐double quadrupole MS. Identity of vitreous proteins was verified by Western blots. The combined total of proteins identified was 255 but only 35 were common to both PVR and control samples, 24 were common to moderate and severe PVR. The regulation model is still a bit murky but should clear soon. Yu, J. et al., Proteomics 2008, 8, 3667–3678.     

Cover Picture: Proteomics 10/2008

In this issue of Proteomics you will find the following highlighted articles: Open‐pit mining for compatible neighbors Open pit mines are an explosive topic in some parts of the US and elsewhere around the world. In this case, however, it is information, not coal or copper, that is being mined from computer files. Ahmad et al. are looking for patterns in the sequence of amino acids that surround a landmark, an amino acid that is frequently modified by phosphorylation or glycosylation. If an appropriate set of rules can be found, it becomes feasible to predict sites of post‐translational modification (PTM) and possibly winners in conflicts from overlapping sites. Algorithms (MAPRes) for O‐glycosylation (GalNAc) and O‐phosphorylation have been implemented that show good fit, correlating well with patterns predicted by existing software. The MAPRes software should also be useful in creating patterns for features such as protease targets and secondary protein structures. Ahmad, I. et al., Proteomics 2008, 8, 1954–1958. Synthetic sequence steals enzyme‐specific (PKCα) spot It is interesting that evolution has optimized, rather than maximized, many interactions. It was only after we maximized these interactions artificially that we began to recognize the subtleties possible with control systems that were not pushed to the max full time. On the other hand, less than 100% is not satisfactory if we are trying to clean out metastasizing tumor cells. Kang et al. are looking for maximum discrimination between protein kinase C (PKC) isozymes for diagnostic and therapeutic applications. PKCα is normally involved in differentiation, growth, and programmed death of many cell types. The researchers began by designing and screening a set of >1700 PKCα target peptides. They selected the one with the highest efficiency of being labeled and characterized it further for kinetics (K_m, and V_max) with 11 PKC isozymes. They also used it for Western blot evaluation of enzyme levels in tumor and normal tissues. Kang, J.‐H. et al., Proteomics 2008, 8, 2006–2011. Subtleties of B. subtilis biological labeling Bacilllus subtilis is a workhorse bacterium, if you'll allow a mixed metaphor. My grad school friends who worked with it always claimed it was a “higher organism” than E. coli because it could differentiate, sort of like yeast. Because it is well studied genetically and physiologically, it has been adopted as a useful model system for the study of stress responses. Dreisbach et al. wanted to extend proteome analysis to membrane proteins under different starvation conditions that generated the stringent response. Conventional methods (e.g. 2‐DE) were not quantitative enough, or had unacceptable error rates (in vitro labelling). They found in vivo labelling with either specific amino acids (SILAC with lysine) or general metabolic labelling (14N/15N‐metabolic) to meet their needs. Samples could be mixed with controls prior to extraction and digestion to markedly reduce technical error rates. Both methods were considered suitable for quantitative proteomic analysis of membrane proteins. Dreisbach, A. et al., Proteomics 2008, 8, 2062–2076.     

In this issue: Proteomics 10/2008

In this issue of Proteomics you will find the following highlighted articles: Open‐pit mining for compatible neighbors Open pit mines are an explosive topic in some parts of the US and elsewhere around the world. In this case, however, it is information, not coal or copper, that is being mined from computer files. Ahmad et al. are looking for patterns in the sequence of amino acids that surround a landmark, an amino acid that is frequently modified by phosphorylation or glycosylation. If an appropriate set of rules can be found, it becomes feasible to predict sites of post‐translational modification (PTM) and possibly winners in conflicts from overlapping sites. Algorithms (MAPRes) for O‐glycosylation (GalNAc) and O‐phosphorylation have been implemented that show good fit, correlating well with patterns predicted by existing software. The MAPRes software should also be useful in creating patterns for features such as protease targets and secondary protein structures. Ahmad, I. et al., Proteomics 2008, 8, 1954–1958. Synthetic sequence steals enzyme‐specific (PKCα) spot It is interesting that evolution has optimized, rather than maximized, many interactions. It was only after we maximized these interactions artificially that we began to recognize the subtleties possible with control systems that were not pushed to the max full time. On the other hand, less than 100% is not satisfactory if we are trying to clean out metastasizing tumor cells. Kang et al. are looking for maximum discrimination between protein kinase C (PKC) isozymes for diagnostic and therapeutic applications. PKCα is normally involved in differentiation, growth, and programmed death of many cell types. The researchers began by designing and screening a set of >1700 PKCα target peptides. They selected the one with the highest efficiency of being labeled and characterized it further for kinetics (K_m, and V_max) with 11 PKC isozymes. They also used it for Western blot evaluation of enzyme levels in tumor and normal tissues. Kang, J.‐H. et al., Proteomics 2008, 8, 2006–2011. Subtleties of B. subtilis biological labeling Bacilllus subtilis is a workhorse bacterium, if you'll allow a mixed metaphor. My grad school friends who worked with it always claimed it was a “higher organism” than E. coli because it could differentiate, sort of like yeast. Because it is well studied genetically and physiologically, it has been adopted as a useful model system for the study of stress responses. Dreisbach et al. wanted to extend proteome analysis to membrane proteins under different starvation conditions that generated the stringent response. Conventional methods (e.g. 2‐DE) were not quantitative enough, or had unacceptable error rates (in vitro labelling). They found in vivo labelling with either specific amino acids (SILAC with lysine) or general metabolic labelling (14N/15N‐metabolic) to meet their needs. Samples could be mixed with controls prior to extraction and digestion to markedly reduce technical error rates. Both methods were considered suitable for quantitative proteomic analysis of membrane proteins. Dreisbach, A. et al., Proteomics 2008, 8, 2062–2076.     

In this issue: Proteomics 8/2008

In this issue of Proteomics you will find the following highlighted articles: Have a heart (mitochondrial) proteome Is a rose always a rose? How clean is clean? Is a proteome always a proteome? Such deep questions to ponder. Zhang et al. don't just ponder, they attack the last two questions. Taking meticulous care to prepare clean mouse cardiac mitochondria, they identify almost a thousand proteins from the functionally and morphologically validated organelle. Half of the proteins had not been previously identified. Functional clusters include the expected and the “under‐appreciated” – proteolysis, protein folding, apoptosis and redox signaling. A close association with rough ER could not be disrupted without damage to the outer mitochondrial membrane. Immunocytological localization of many of the proteins revealed roles in other sites as well, including ER, cytoplasm, and Golgi. Comparative analysis of published mitochondrial proteomes from different tissues suggests that the proteomes are functionally adapted to their particular milieu. A mitochondrion (heart) is not a mitochondrion (liver). Zhang, J. et al., Proteomics 2008, 8, 1564–1575. Ibuprofen: split personality complicates proteome analyses Ibuprofen is one of those two‐fisted drugs that comes in an S form and an R form. The S form of this nonsteroidal anti‐inflammatory drug (NSAID) is the only active one, in this case. Normally sold over the counter for general aches and pains in the US, statistical analysis of its regular users has found it associated with a reduced incidence of Alzheimer's disease. Following up on this lead, Zhang et al. performed proteomic analysis of the effect of the R and S forms and their mixture on neuroblastoma cells. From three replicates, 167 proteins were identified as being quantitatively shifted. A total of 13 were unique. Functionally, they included representatives from metabolic enzymes (5), signaling (6), and cytoskeleton (2). Of interest for the Alzheimer's association was the reduced levels of reactive oxygen species (ROS), probably linked to levels of peroxiredoxins 2 and 6 in ibuprofen S‐treated cells. Zhang, J. et al., Proteomics 2008, 8, 1595–1607. Not your usual marine bacterium Rhodopirellula baltica is a member of the Planctomycetes phylum. These bacteria exhibit a proteinaceous cell wall, budding cell division, and intracellular compartments. From genome sequencing, it has >7300 ORFs. Analyzing the soluble proteins over the range of pH 3–10 by 2‐D PAGE, using narrow range pH gradient gels, nHPLC‐MS, and 1‐D SDS‐PAGE, Hieu et al. added 709 proteins to the proteins identified previously to bring the total identified to 1267, 17% of the predicted total ORFs. Gel‐free analysis (multiple dimension LC‐MS) yielded 145 proteins not seen in gel‐based methods. Both 1‐D and gel‐free methods were used for identification of cell wall and ribosomal proteins. Ninety three proteins were identified in the cell wall proteome and 13 extracellular proteins. No support was found for the hypothesis that R. baltica fed on sinking dead “marine snow” organisms by secreting proteases. Hieu, C. X. et al., Proteomics 2008, 8, 1608–1623.     

Cover Picture: Proteomics 8/2008

In this issue of Proteomics you will find the following highlighted articles: Have a heart (mitochondrial) proteome Is a rose always a rose? How clean is clean? Is a proteome always a proteome? Such deep questions to ponder. Zhang et al. don't just ponder, they attack the last two questions. Taking meticulous care to prepare clean mouse cardiac mitochondria, they identify almost a thousand proteins from the functionally and morphologically validated organelle. Half of the proteins had not been previously identified. Functional clusters include the expected and the “under‐appreciated” – proteolysis, protein folding, apoptosis and redox signaling. A close association with rough ER could not be disrupted without damage to the outer mitochondrial membrane. Immunocytological localization of many of the proteins revealed roles in other sites as well, including ER, cytoplasm, and Golgi. Comparative analysis of published mitochondrial proteomes from different tissues suggests that the proteomes are functionally adapted to their particular milieu. A mitochondrion (heart) is not a mitochondrion (liver). Zhang, J. et al., Proteomics 2008, 8, 1564–1575. Ibuprofen: split personality complicates proteome analyses Ibuprofen is one of those two‐fisted drugs that comes in an S form and an R form. The S form of this nonsteroidal anti‐inflammatory drug (NSAID) is the only active one, in this case. Normally sold over the counter for general aches and pains in the US, statistical analysis of its regular users has found it associated with a reduced incidence of Alzheimer's disease. Following up on this lead, Zhang et al. performed proteomic analysis of the effect of the R and S forms and their mixture on neuroblastoma cells. From three replicates, 167 proteins were identified as being quantitatively shifted. A total of 13 were unique. Functionally, they included representatives from metabolic enzymes (5), signaling (6), and cytoskeleton (2). Of interest for the Alzheimer's association was the reduced levels of reactive oxygen species (ROS), probably linked to levels of peroxiredoxins 2 and 6 in ibuprofen S‐treated cells. Zhang, J. et al., Proteomics 2008, 8, 1595–1607. Not your usual marine bacterium Rhodopirellula baltica is a member of the Planctomycetes phylum. These bacteria exhibit a proteinaceous cell wall, budding cell division, and intracellular compartments. From genome sequencing, it has >7300 ORFs. Analyzing the soluble proteins over the range of pH 3–10 by 2‐D PAGE, using narrow range pH gradient gels, nHPLC‐MS, and 1‐D SDS‐PAGE, Hieu et al. added 709 proteins to the proteins identified previously to bring the total identified to 1267, 17% of the predicted total ORFs. Gel‐free analysis (multiple dimension LC‐MS) yielded 145 proteins not seen in gel‐based methods. Both 1‐D and gel‐free methods were used for identification of cell wall and ribosomal proteins. Ninety three proteins were identified in the cell wall proteome and 13 extracellular proteins. No support was found for the hypothesis that R. baltica fed on sinking dead “marine snow” organisms by secreting proteases. Hieu, C. X. et al., Proteomics 2008, 8, 1608–1623.     

Cover Picture: Proteomics 13/2008

In this issue of Proteomics you will find the following highlighted articles: Mini pig kidney pie? A lot of antigens to chew on Miniature pigs have been of interest as potential organ xeno‐transplant donors for a number of years but mostly without success. A galactosyl transferase gene knock‐out heart lasted for 6 months, but then succumbed to vascular rejection, indicating previously unrecognized antigens. Kim, et al. applied current glycome analysis techniques to mini‐pig kidney surface antigens. They found an abundance of new ones–over 100 N‐glycans total, some sialylated, some neutral, some never reported before. The structures of many were determined and relatively quantitated. What was sauce for the kidney was not necessarily sauce for the heart. The information gathered and the questions raised will keep transplanters chewing for a long time. Y.‐G. Kim et al., Proteomics 2008, 8, 2596–2610. PACE‐ing along with the DUKX that are really hamsters Turning a marching band or moving it through a bottleneck requires different speeds at different points across the ranks. So does maximal production of biologically produced pharmaceuticals. Here Meleady, et al. use 2‐D DIGE technology to look at the required proteins and the levels of expression required for optimal production of human bone morphogenetic protein 2 (rhBMP‐2) in Chinese hamster ovary‐derived cell lines (CHO DUKX and engineered derivatives). Maturation of BMP‐2 requires the action of PACE (paired basic amino acid cleaving enzyme) and PACE levels are improved by co‐transfection with a soluble PACE gene. With high levels of PACE activity, yields of BMP‐2 improved 4‐fold. PACEsol enhances production of a variety of other proteins as well. Comparison of DUKX‐BMP‐2 cells expressing vs. not expressing PACEsol showed ～180 differentially expressed proteins, 60 identified, that were assigned to a number of functional categories. P. Meleady et al., Proteomics 2008, 8, 2611–2624. Ever deeper into cheesy secretome Kluyveromyces lactis, a budding yeast related to Saccharomyces cerevisiae, is of genetic and industrial interest. Its name comes from its ability to convert sweet milk to sour by fermentation of lactose to lactic acid, not quite the same as glucose to ethanol, but useful nonetheless. Industrially, it has been engineered to produce a vegetarian rennet for cheese‐making as well as other secreted protein products. Swaim, et al. compared the proteins in spent fermentation broth of the industrial expression strain K. lactis GG799 to the predicted secretion products based on genome sequence information and to predicted secretions from Candida albicans and S. cerevisiae. Using multidimensional LC‐MS/MS to analyze tryptic digests, they found 81 secreted products out of 178 predicted. Twenty‐six of those did not exhibit an N‐terminal secretion signal, suggesting that there are alternative pathways to the cell surface. An intracellular nano‐Swiss, perhaps? C. L. Swaim et al., Proteomics 2008, 8, 2714–2723.     

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 13/2008

In this issue of Proteomics you will find the following highlighted articles: Mini pig kidney pie? A lot of antigens to chew on Miniature pigs have been of interest as potential organ xeno‐transplant donors for a number of years but mostly without success. A galactosyl transferase gene knock‐out heart lasted for 6 months, but then succumbed to vascular rejection, indicating previously unrecognized antigens. Kim, et al. applied current glycome analysis techniques to mini‐pig kidney surface antigens. They found an abundance of new ones–over 100 N‐glycans total, some sialylated, some neutral, some never reported before. The structures of many were determined and relatively quantitated. What was sauce for the kidney was not necessarily sauce for the heart. The information gathered and the questions raised will keep transplanters chewing for a long time. Y.‐G. Kim et al., Proteomics 2008, 8, 2596–2610. PACE‐ing along with the DUKX that are really hamsters Turning a marching band or moving it through a bottleneck requires different speeds at different points across the ranks. So does maximal production of biologically produced pharmaceuticals. Here Meleady, et al. use 2‐D DIGE technology to look at the required proteins and the levels of expression required for optimal production of human bone morphogenetic protein 2 (rhBMP‐2) in Chinese hamster ovary‐derived cell lines (CHO DUKX and engineered derivatives). Maturation of BMP‐2 requires the action of PACE (paired basic amino acid cleaving enzyme) and PACE levels are improved by co‐transfection with a soluble PACE gene. With high levels of PACE activity, yields of BMP‐2 improved 4‐fold. PACEsol enhances production of a variety of other proteins as well. Comparison of DUKX‐BMP‐2 cells expressing vs. not expressing PACEsol showed ～180 differentially expressed proteins, 60 identified, that were assigned to a number of functional categories. P. Meleady et al., Proteomics 2008, 8, 2611–2624. Ever deeper into cheesy secretome Kluyveromyces lactis, a budding yeast related to Saccharomyces cerevisiae, is of genetic and industrial interest. Its name comes from its ability to convert sweet milk to sour by fermentation of lactose to lactic acid, not quite the same as glucose to ethanol, but useful nonetheless. Industrially, it has been engineered to produce a vegetarian rennet for cheese‐making as well as other secreted protein products. Swaim, et al. compared the proteins in spent fermentation broth of the industrial expression strain K. lactis GG799 to the predicted secretion products based on genome sequence information and to predicted secretions from Candida albicans and S. cerevisiae. Using multidimensional LC‐MS/MS to analyze tryptic digests, they found 81 secreted products out of 178 predicted. Twenty‐six of those did not exhibit an N‐terminal secretion signal, suggesting that there are alternative pathways to the cell surface. An intracellular nano‐Swiss, perhaps? C. L. Swaim et al., Proteomics 2008, 8, 2714–2723.     

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.     

Cover Picture: Proteomics 15/2008

In this issue of Proteomics you will find the following highlighted articles: An old dog refines new tricks Old dogs are reputed to be slow learners but they can be subtle manipulators, able to induce younger dogs and gullible owners to share the food dish in their favor or choose the path they prefer. Two‐dimensional gel electrophoresis has been around for more than 25 years but “new and improved” versions continue to appear. Ericsson et al. scramble the order of several steps to get more information out of the combination of IPG/IEF and “shotgun” peptide analysis. Developed for studying mechanisms of drug resistance in small cell lung cancer, the modified protocol fractionates sonically disrupted cells into microsomes and soluble fractions before tryptic digestion and iTRAQ labeling for later quantitation. Digested samples were fractionated on narrow range immobilized pH gradient strips from which they were eluted for MALDI TOF or LC‐MS/MS analysis. Detection and identification of transmembrane proteins were dramatically improved. Ericsson, H. et al., Proteomics 2008, 8, 3008–3018. An evanescent view of a lectin micro‐array: through a glass faintly Lectins have the ability to distinguish closely‐related carbohydrate moieties attached (or not) to other molecules such as proteins, peptides, lipids, cells, etc. In some respects, they are much like antibodies, just not quite as specific. Using an array of 45 different lectins, the glycan portion of glycoproteins can be identified by its binding profile. Here, Uchiyama et al. report the improvements they have made to the reproducibility and sensitivity of the system. The binding of rhodamine‐labeled probes was detected in an evanescent field fluorescence‐based instrument that was capable of reaching, 10pM levels without having to wash off unbound probe. Depositing lectin spots with a non‐contact type printer, at the right humidity, and blocking with a non‐proteinaceous material greatly improved sensitivity. Uchiyama, N. et al., Proteomics 2008, 8, 3042–3050. The innate defense: multiplex proteomic probing The body's first line of defense against pathogen infection is the innate response. In the case of bacterial infection, it is initiated by the sensing of the universal Gram‐positive cell wall lipopolysaccharide (LPS) component by macrophages primarily (but not solely) through the Toll‐like receptor 4 (TLR4). To understand the regulation of the LPS response, Gu et al. developed a multiplex quantitative proteomic analysis procedure to follow the response of TLR4+ and TLR4? cell lines. The method of choice was amino acid‐coded mass tagging (AACT, also referred to as SILAC). It showed high efficiency of labeling (95%) which eliminated interference with quantitation by the unlabeled fraction. Using triplex labeling of lysine (13C, 15N), the authors confirmed that TLR4? cells did show a response to LPS: 25 proteins were up‐regulated in TLR4+ cells, 5 in TLR4? cells. More than 500 proteins could be quantitated. Gu, S. et al., Proteomics 2008, 8, 3061–3070.     

In this issue: Proteomics 15/2008

In this issue of Proteomics you will find the following highlighted articles: An old dog refines new tricks Old dogs are reputed to be slow learners but they can be subtle manipulators, able to induce younger dogs and gullible owners to share the food dish in their favor or choose the path they prefer. Two‐dimensional gel electrophoresis has been around for more than 25 years but “new and improved” versions continue to appear. Ericsson et al. scramble the order of several steps to get more information out of the combination of IPG/IEF and “shotgun” peptide analysis. Developed for studying mechanisms of drug resistance in small cell lung cancer, the modified protocol fractionates sonically disrupted cells into microsomes and soluble fractions before tryptic digestion and iTRAQ labeling for later quantitation. Digested samples were fractionated on narrow range immobilized pH gradient strips from which they were eluted for MALDI TOF or LC‐MS/MS analysis. Detection and identification of transmembrane proteins were dramatically improved. Ericsson, H. et al., Proteomics 2008, 8, 3008–3018. An evanescent view of a lectin micro‐array: through a glass faintly Lectins have the ability to distinguish closely‐related carbohydrate moieties attached (or not) to other molecules such as proteins, peptides, lipids, cells, etc. In some respects, they are much like antibodies, just not quite as specific. Using an array of 45 different lectins, the glycan portion of glycoproteins can be identified by its binding profile. Here, Uchiyama et al. report the improvements they have made to the reproducibility and sensitivity of the system. The binding of rhodamine‐labeled probes was detected in an evanescent field fluorescence‐based instrument that was capable of reaching, 10pM levels without having to wash off unbound probe. Depositing lectin spots with a non‐contact type printer, at the right humidity, and blocking with a non‐proteinaceous material greatly improved sensitivity. Uchiyama, N. et al., Proteomics 2008, 8, 3042–3050. The innate defense: multiplex proteomic probing The body's first line of defense against pathogen infection is the innate response. In the case of bacterial infection, it is initiated by the sensing of the universal Gram‐positive cell wall lipopolysaccharide (LPS) component by macrophages primarily (but not solely) through the Toll‐like receptor 4 (TLR4). To understand the regulation of the LPS response, Gu et al. developed a multiplex quantitative proteomic analysis procedure to follow the response of TLR4+ and TLR4? cell lines. The method of choice was amino acid‐coded mass tagging (AACT, also referred to as SILAC). It showed high efficiency of labeling (95%) which eliminated interference with quantitation by the unlabeled fraction. Using triplex labeling of lysine (13C, 15N), the authors confirmed that TLR4? cells did show a response to LPS: 25 proteins were up‐regulated in TLR4+ cells, 5 in TLR4? cells. More than 500 proteins could be quantitated. Gu, S. et al., Proteomics 2008, 8, 3061–3070.     

Cover Picture: Proteomics 14/2008

In this issue of Proteomics you will find the following highlighted articles: A spoonful of reality helps the errors go down Physicists predict particles from a theory then go hunting for them. To see if they fit the theory, the scientists make measurements of mass, spin, half‐life, charge, symmetry, etc. Life scientists tend to find particles first, then theorize about what they found. Martinez et al. have developed pattern search/match software that is trained on a set of known data (amino acid sequences) to recognize signal sequences that cause cellular enzymes to modify N‐termini of specified cytosolic proteins. Their accuracy rose dramatically when they expanded the amount of training material and segregated it into modules for the various kingdoms (archaea, fungi, plants, animals, eubacteria). Terminal modifications examined included presence or absence of initial methionine, N‐acetylation, N‐myristoylation, and S‐palmitoylation. The particular modification led to varying degrees of internal accumulation. Martinez, A. et al., Proteomics 2008, 8, 2809–2831. Please don't pet the fish Salmon appear to have no fear of jumping several decimeters out of water on their annual upstream migration but they do seem to have a fear of being lifted out of water for a few seconds once a day. Liu et al. looked at the degree of O‐acetylation of serum N‐glycans of Atlantic salmon over a period of 4 weeks. The salmon N‐glycan pattern was similar to the human. Stress was created by holding the fish out of water for 15 seconds daily. Stressed fish showed a reduced level of mono‐O‐acetylated sialic acids. Di‐O‐acetylated species increased, however, over the 4‐week experimental period. The increase in O‐acetylsialic acid is a potential biomarker for long‐term stress in fish. More work is needed to evaluate the extensibility of these findings. Liu, X. et al., Proteomics 2008, 8, 2849–2857. Cattle and ART and proteomics The ART of cattle raising is not learned in a Parson's School of Bovine Design or Parisian cow ecole, it is “Assisted Reproduction Technologies” and is taught in veterinary programs and schools of agriculture. It includes in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT), and multiple ovulation embryo transfer. Expensive procedures requiring specialized training, they are normally applied only to genetically superior breeding stock. Fetal losses are high — 2× to >10× natural fertilization failure rates. Riding et al. used proteomic technology (2‐D DIGE, MALDI‐TOF‐MS/MS) to look for biomarkers in conceptus fluids. In particular, they sought indicators of fetal‐maternal environment status and fetal health at 45 and 90 days post‐conception for IVF and SCNT. Allantoic fluid samples from 45 days showed elevated levels of cathelicidin antimicrobial protein (CAMP) family members (3 of 4 IVF, up ≤100×; 2 of 4 SCNT, up ≤45×; natural, up ≤6×) and several other proteins (PGLYRP, SERPINB1, COLT1). Riding, G. A. et al., Proteomics 2008, 8, 2967–2982.     

In this issue: Proteomics 14/2008

In this issue of Proteomics you will find the following highlighted articles: A spoonful of reality helps the errors go down Physicists predict particles from a theory then go hunting for them. To see if they fit the theory, the scientists make measurements of mass, spin, half‐life, charge, symmetry, etc. Life scientists tend to find particles first, then theorize about what they found. Martinez et al. have developed pattern search/match software that is trained on a set of known data (amino acid sequences) to recognize signal sequences that cause cellular enzymes to modify N‐termini of specified cytosolic proteins. Their accuracy rose dramatically when they expanded the amount of training material and segregated it into modules for the various kingdoms (archaea, fungi, plants, animals, eubacteria). Terminal modifications examined included presence or absence of initial methionine, N‐acetylation, N‐myristoylation, and S‐palmitoylation. The particular modification led to varying degrees of internal accumulation. Martinez, A. et al., Proteomics 2008, 8, 2809–2831. Please don't pet the fish Salmon appear to have no fear of jumping several decimeters out of water on their annual upstream migration but they do seem to have a fear of being lifted out of water for a few seconds once a day. Liu et al. looked at the degree of O‐acetylation of serum N‐glycans of Atlantic salmon over a period of 4 weeks. The salmon N‐glycan pattern was similar to the human. Stress was created by holding the fish out of water for 15 seconds daily. Stressed fish showed a reduced level of mono‐O‐acetylated sialic acids. Di‐O‐acetylated species increased, however, over the 4‐week experimental period. The increase in O‐acetylsialic acid is a potential biomarker for long‐term stress in fish. More work is needed to evaluate the extensibility of these findings. Liu, X. et al., Proteomics 2008, 8, 2849–2857. Cattle and ART and proteomics The ART of cattle raising is not learned in a Parson's School of Bovine Design or Parisian cow ecole, it is “Assisted Reproduction Technologies” and is taught in veterinary programs and schools of agriculture. It includes in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT), and multiple ovulation embryo transfer. Expensive procedures requiring specialized training, they are normally applied only to genetically superior breeding stock. Fetal losses are high — 2× to >10× natural fertilization failure rates. Riding et al. used proteomic technology (2‐D DIGE, MALDI‐TOF‐MS/MS) to look for biomarkers in conceptus fluids. In particular, they sought indicators of fetal‐maternal environment status and fetal health at 45 and 90 days post‐conception for IVF and SCNT. Allantoic fluid samples from 45 days showed elevated levels of cathelicidin antimicrobial protein (CAMP) family members (3 of 4 IVF, up ≤100×; 2 of 4 SCNT, up ≤45×; natural, up ≤6×) and several other proteins (PGLYRP, SERPINB1, COLT1). Riding, G. A. et al., Proteomics 2008, 8, 2967–2982.     

Cover Picture: Proteomics 22/2008

In this issue of Proteomics you will find the following highlighted articles: Man bites dog! Noise improves signal! Yes, the right kind of noise does improve the signal (by about 10‐fold in the LC/MS case described here). Scheltema et al. used the noise generated by the ions remaining in the sample from the LC step as internal standards to standardize and calibrate the mass spectrum of interest. Given a set of well characterized contaminants at very low, but detectable levels, the researchers were able to appropriately stretch or compress spectra by comparison to a reference spectrum of contaminants expected in a particular sample. The demonstration was performed on a Thermo Fisher LTQ Orbitrap system which, run conventionally, yielded a mass accuracy of 1 to 2 parts per million. When the noise method was applied to the same data, the mass accuracy was 0.21 ppm. Scheltema, R. A. et al., Proteomics 2008, 8, 4647–4656. Rafting down the Melanoma river When the subject is rafts, Mark Twain's story of Tom Sawyer and Huckleberry Finn rafting down the Mississippi comes immediately to mind for most Americans. A raft of interest to life scientists is associated with detergent resistant membranes found in malignant melanoma cell lines. Made of predominantly cholesterol and sphingolipids, the raft and associated proteins have been shown to participate in signal regulation and protein trafficking as well as several diseases. Working from this information, Baruthio et al. have looked at the lipid raft proteome as a function of melanoma malignancy stage using LC‐MS/MS: radial growth phase, (pre‐metastatic); early vertical growth phase, (non‐metastatic); and fully transformed. They found >175 proteins total in all stages, the most abundant was AHNAK, a large membrane protein. Groups of potential stage markers were detected, although with some difficulty in reproducibility of extraction. Functions found included vacuolar ATPases, adhesion molecules, and signaling pathway regulators. Baruthio, F. et al., Proteomics 2008, 8, 4733–4747. Hot peppers maker confusing soup Capsaicin is the naturally occurring compound that gives chili peppers their “heat.” It is also a component of the pepper's arsenal, deterring some types of attacks. Another of its roles is in regulation of programmed cell death, apoptosis: sometimes it promotes it, sometimes it inhibits it and it always seems to involve reactive oxygen species (ROS). To look at its function as a potential anti‐cancer agent, Baek et al. compared its effect on two human cancer cell lines. HepG2, a hepatoblastoma and SK‐N‐SH, a neuroblastoma, were examined for proteomic changes after exposure to capsaicin at various levels and for various times. Both blastomas responded but in markedly different fashions. Apoptosis was induced in both cell lines, but the ROS levels were up in HepG2 and down in SK‐N‐SH. A number of ROS enzymes exhibited anomalous expression level changes, possibly due to the number of enzymes involved. Baek, Y. M. et al., Proteomics 2008, 8, 4748–4767.     

In this issue: Proteomics 22/2008

In this issue of Proteomics you will find the following highlighted articles: Man bites dog! Noise improves signal! Yes, the right kind of noise does improve the signal (by about 10‐fold in the LC/MS case described here). Scheltema et al. used the noise generated by the ions remaining in the sample from the LC step as internal standards to standardize and calibrate the mass spectrum of interest. Given a set of well characterized contaminants at very low, but detectable levels, the researchers were able to appropriately stretch or compress spectra by comparison to a reference spectrum of contaminants expected in a particular sample. The demonstration was performed on a Thermo Fisher LTQ Orbitrap system which, run conventionally, yielded a mass accuracy of 1 to 2 parts per million. When the noise method was applied to the same data, the mass accuracy was 0.21 ppm. Scheltema, R. A. et al., Proteomics 2008, 8, 4647–4656. Rafting down the Melanoma river When the subject is rafts, Mark Twain's story of Tom Sawyer and Huckleberry Finn rafting down the Mississippi comes immediately to mind for most Americans. A raft of interest to life scientists is associated with detergent resistant membranes found in malignant melanoma cell lines. Made of predominantly cholesterol and sphingolipids, the raft and associated proteins have been shown to participate in signal regulation and protein trafficking as well as several diseases. Working from this information, Baruthio et al. have looked at the lipid raft proteome as a function of melanoma malignancy stage using LC‐MS/MS: radial growth phase, (pre‐metastatic); early vertical growth phase, (non‐metastatic); and fully transformed. They found >175 proteins total in all stages, the most abundant was AHNAK, a large membrane protein. Groups of potential stage markers were detected, although with some difficulty in reproducibility of extraction. Functions found included vacuolar ATPases, adhesion molecules, and signaling pathway regulators. Baruthio, F. et al., Proteomics 2008, 8, 4733–4747. Hot peppers maker confusing soup Capsaicin is the naturally occurring compound that gives chili peppers their “heat.” It is also a component of the pepper's arsenal, deterring some types of attacks. Another of its roles is in regulation of programmed cell death, apoptosis: sometimes it promotes it, sometimes it inhibits it and it always seems to involve reactive oxygen species (ROS). To look at its function as a potential anti‐cancer agent, Baek et al. compared its effect on two human cancer cell lines. HepG2, a hepatoblastoma and SK‐N‐SH, a neuroblastoma, were examined for proteomic changes after exposure to capsaicin at various levels and for various times. Both blastomas responded but in markedly different fashions. Apoptosis was induced in both cell lines, but the ROS levels were up in HepG2 and down in SK‐N‐SH. A number of ROS enzymes exhibited anomalous expression level changes, possibly due to the number of enzymes involved. Baek, Y. M. et al., Proteomics 2008, 8, 4748–4767.