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.     

In this issue: Proteomics 7/2008

In this issue of Proteomics you will find the following highlighted articles: Modified amino peptides step out of line, reveal identity In thriller movies and spy stories, you can often tell which character is a bad guy if his “confession” changes under pressure or depends on the inquisitor. Likewise for peptides with modifications. Staes et al. use a similar technique to find α‐amino blocked peptides. After chromatography of a digest over a C₁₈ reverse phase column, fractions were treated with TNBS and re‐chromatographed on the same column, under the same conditions. The peptides that had trypsin‐exposed amino groups became much more hydrophobic in the second round because of the addition of the TNBS. The technique (COFRADIC) was also improved by preceding the C₁₈ column by use of a strong cation exchange for fractionation and using a kit for removal of any pyrrolidone carboxylic acid termini from peptides. The revised protocol raised the yield of true amino termini from 60% to 95%. Staes, A. et al., Proteomics 2008, 8, 1362–1370. Decrypting Cryptosporidium parvum: Proteome data revealed by triple analysis As hikers in North America and normal people in many parts of the world know, Cryptosporidium parvum is a protozoan parasite that causes an unpleasant intestinal infection in humans. It also infects livestock species, which leads to widespread waterborne transmission unless effective water treatment is employed. When the oocytes enter the gastrointestinal tract, they are stimulated to undergo excystation, releasing four sporozoites that enter the epithelial cells. There they undergo asexual reproduction and begin a complex series of steps before reproduction is complete and oocytes are released. Although the genome has been completely sequenced, many of the proteins predicted did not have recognizable functions. Sanderson et al. used a tissue culture system of excystation to collect enough sporozoites for proteomic analysis by MuDPIT and LC‐MS/MS after (a) 2‐DE and (b) 1‐DE. Over 1200 unique proteins were identified, representing >30% of the predicted organism proteome, >200 of which had transmembrane domains. Sanderson, S. J. et al., Proteomics 2008, 8, 1398–1414. Oxidized proteins in serum: Inside job or outside contractor? Reactive oxygen species (ROS) seem to be involved in a variety of diseases, including Alzheimer's, Parkinson's, cancer and heart disease. Searches for biomarkers for these diseases have most commonly been done in blood plasma, which contains proteins from essentially every cell type and tissue in the organism. Mirzaei et al. explore questions of cause and effect in rat plasma by trapping ROS‐caused carbonylation points with biotin hydrazide, followed by avidin affinity chromatography and proteomic analysis (LC‐MS/MS). Of 146 proteins identified in four rats, 44 had at least one carbonylation site and 38 had two or more sites. Over 30% of the proteins were membrane proteins, suggesting a major source of ROS was external, a hypothesis supported by the observation that mitochondrial proteins are not affected, despite their proximity to endogenous ROS. On the other hand, 13% were nuclear proteins. Another surprise: virtually no (2%) plasma proteins were found. Mirzaei, H. et al., Proteomics 2008, 8, 1516–1527.     

Cover Picture: Proteomics 7/2008

In this issue of Proteomics you will find the following highlighted articles: Modified amino peptides step out of line, reveal identity In thriller movies and spy stories, you can often tell which character is a bad guy if his “confession” changes under pressure or depends on the inquisitor. Likewise for peptides with modifications. Staes et al. use a similar technique to find α‐amino blocked peptides. After chromatography of a digest over a C₁₈ reverse phase column, fractions were treated with TNBS and re‐chromatographed on the same column, under the same conditions. The peptides that had trypsin‐exposed amino groups became much more hydrophobic in the second round because of the addition of the TNBS. The technique (COFRADIC) was also improved by preceding the C₁₈ column by use of a strong cation exchange for fractionation and using a kit for removal of any pyrrolidone carboxylic acid termini from peptides. The revised protocol raised the yield of true amino termini from 60% to 95%. Staes, A. et al., Proteomics 2008, 8, 1362–1370. Decrypting Cryptosporidium parvum: Proteome data revealed by triple analysis As hikers in North America and normal people in many parts of the world know, Cryptosporidium parvum is a protozoan parasite that causes an unpleasant intestinal infection in humans. It also infects livestock species, which leads to widespread waterborne transmission unless effective water treatment is employed. When the oocytes enter the gastrointestinal tract, they are stimulated to undergo excystation, releasing four sporozoites that enter the epithelial cells. There they undergo asexual reproduction and begin a complex series of steps before reproduction is complete and oocytes are released. Although the genome has been completely sequenced, many of the proteins predicted did not have recognizable functions. Sanderson et al. used a tissue culture system of excystation to collect enough sporozoites for proteomic analysis by MuDPIT and LC‐MS/MS after (a) 2‐DE and (b) 1‐DE. Over 1200 unique proteins were identified, representing >30% of the predicted organism proteome, >200 of which had transmembrane domains. Sanderson, S. J. et al., Proteomics 2008, 8, 1398–1414. Oxidized proteins in serum: Inside job or outside contractor? Reactive oxygen species (ROS) seem to be involved in a variety of diseases, including Alzheimer's, Parkinson's, cancer and heart disease. Searches for biomarkers for these diseases have most commonly been done in blood plasma, which contains proteins from essentially every cell type and tissue in the organism. Mirzaei et al. explore questions of cause and effect in rat plasma by trapping ROS‐caused carbonylation points with biotin hydrazide, followed by avidin affinity chromatography and proteomic analysis (LC‐MS/MS). Of 146 proteins identified in four rats, 44 had at least one carbonylation site and 38 had two or more sites. Over 30% of the proteins were membrane proteins, suggesting a major source of ROS was external, a hypothesis supported by the observation that mitochondrial proteins are not affected, despite their proximity to endogenous ROS. On the other hand, 13% were nuclear proteins. Another surprise: virtually no (2%) plasma proteins were found. Mirzaei, H. et al., Proteomics 2008, 8, 1516–1527.     

Cover Picture: Proteomics 11/2008

In this issue of Proteomics you will find the following highlighted articles: Pancreatic cancer signs autograph on micro antibody array Pancreatic cancer has been one of the nastier members of the “Discovered‐too‐late‐to‐do‐anything‐about‐it” disease club. Its 5‐year survival rate is 3–5 % because of late diagnosis and no effective therapy for advanced disease cases. This paper by Ingvarsson et al. reports their encouraging findings on the use of recombinant antibody microarrays to survey serum for diagnostic and prognostic proteins. In these “proof‐of‐concept” experiments they found a signature of 19 unique scFv antibodies, specific for immunoregulatory proteins, that could distinguish pancreatic cancer from normal and from Helicobacter pylori (an indicator of inflammation, 3 out of 14 overlap). The test panel distinguished long and short survivors (with only one long survivor misclassified). Data was classified using a Support Vector Machine. The classifier was validated by multiple splits of the data and leave‐one‐out tests. Ingvarsson, J. et al., Proteomics 2008, 8, 2211–2219. Of cadmium and zinc: Brothers or not? Cadmium and zinc occupy the same column in the periodic table so you might expect some biological similarities. Not much luck – mercury is also in that column. Zinc, under tight control, is an essential mineral; cadmium is toxic and induces a variety of defensive responses. A highly zinc‐resistant cell line (HZR) has been derived from the human HeLa line. Rousselet et al. have compared the proteomes of HZR and HeLa cultured in Cd and Zn using a variety of proteomic and genomic tools. MALDI‐TOF MS after 2‐DE revealed examples of a co‐chaperone, a heat‐shock organizing protein (Hop), ubiquitin and a number of reactive oxygen species control proteins elevated in HZR. Of special interest was 4‐hydroxyphenyl‐pyruvate dioxygenase (HPPD), catalyst of one of the first breakdown steps of tyrosine. The complex relationships revealed will require a lot more than one paragraph for explanation. Rousselet, E. et al., Proteomics 2008, 8, 2244–2255. Grey box proteomics of salty species In the classic black box experiment you know nothing about the contents of the box. I propose a grey box for experiments directed by homologous knowledge – like these. Pandhal et al. have developed a protocol for proteomic analysis of an unsequenced species by homology. The organism of interest is a halotolerant cyanobacterium, Euhalothece sp. which can grow in NaCl concentrations ranging from 0% to >9% NaCl. The nearest sequenced relative is a Synechocystis sp. By metabolic labeling with ¹⁵N/­¹⁴N, the researchers were able to use MS to match proteins from the two species and also quantitate changes in levels of proteins in response to salt levels. Three labelling experiments ([% NaCl], 0% +3%, 3% +6%, and 3% +9%) yielded 229, 212, and 96 proteins, respectively, by MASCOT search of proteins with two peptides of each isotope. MS BLAST found 32, 30, and 7 more proteins, respectively. Pandhal, J. et al., Proteomics 2008, 8, 2266–2284.     

In this issue: Proteomics 11/2008

In this issue of Proteomics you will find the following highlighted articles: Pancreatic cancer signs autograph on micro antibody array Pancreatic cancer has been one of the nastier members of the “Discovered‐too‐late‐to‐do‐anything‐about‐it” disease club. Its 5‐year survival rate is 3–5 % because of late diagnosis and no effective therapy for advanced disease cases. This paper by Ingvarsson et al. reports their encouraging findings on the use of recombinant antibody microarrays to survey serum for diagnostic and prognostic proteins. In these “proof‐of‐concept” experiments they found a signature of 19 unique scFv antibodies, specific for immunoregulatory proteins, that could distinguish pancreatic cancer from normal and from Helicobacter pylori (an indicator of inflammation, 3 out of 14 overlap). The test panel distinguished long and short survivors (with only one long survivor misclassified). Data was classified using a Support Vector Machine. The classifier was validated by multiple splits of the data and leave‐one‐out tests. Ingvarsson, J. et al., Proteomics 2008, 8, 2211–2219. Of cadmium and zinc: Brothers or not? Cadmium and zinc occupy the same column in the periodic table so you might expect some biological similarities. Not much luck – mercury is also in that column. Zinc, under tight control, is an essential mineral; cadmium is toxic and induces a variety of defensive responses. A highly zinc‐resistant cell line (HZR) has been derived from the human HeLa line. Rousselet et al. have compared the proteomes of HZR and HeLa cultured in Cd and Zn using a variety of proteomic and genomic tools. MALDI‐TOF MS after 2‐DE revealed examples of a co‐chaperone, a heat‐shock organizing protein (Hop), ubiquitin and a number of reactive oxygen species control proteins elevated in HZR. Of special interest was 4‐hydroxyphenyl‐pyruvate dioxygenase (HPPD), catalyst of one of the first breakdown steps of tyrosine. The complex relationships revealed will require a lot more than one paragraph for explanation. Rousselet, E. et al., Proteomics 2008, 8, 2244–2255. Grey box proteomics of salty species In the classic black box experiment you know nothing about the contents of the box. I propose a grey box for experiments directed by homologous knowledge – like these. Pandhal et al. have developed a protocol for proteomic analysis of an unsequenced species by homology. The organism of interest is a halotolerant cyanobacterium, Euhalothece sp. which can grow in NaCl concentrations ranging from 0% to >9% NaCl. The nearest sequenced relative is a Synechocystis sp. By metabolic labeling with ¹⁵N/­¹⁴N, the researchers were able to use MS to match proteins from the two species and also quantitate changes in levels of proteins in response to salt levels. Three labelling experiments ([% NaCl], 0% +3%, 3% +6%, and 3% +9%) yielded 229, 212, and 96 proteins, respectively, by MASCOT search of proteins with two peptides of each isotope. MS BLAST found 32, 30, and 7 more proteins, respectively. Pandhal, J. et al., Proteomics 2008, 8, 2266–2284.     

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.     

In this issue: Proteomics 9/2009

In this issue of Proteomics you will find the following highlighted articles: Rafting on the pond It seems that any river with a drop of more than 20‐30 cm/km is a candidate for a commercially viable rafting business. Biochemical rafters are pickier. They need a detergent‐resistant lipid raft where they can set up their signaling system. Kim et al. examined the changes in the raft molecules involved in insulin stimulated pre‐adipocyte to adipocyte differentiation (adipogenesis). A substantial number of adipocyte raft‐specific proteins were identified by immunoblots and confirmed by 2‐DE MS. A protein of particular interest was gC1qR, specific for mature adipocyte rafts, which also binds complement C1q and a number of other extracellular proteins (vitronectin, fibrinogen, hyaluronic acids . . .). Down‐regulation of gC1qR by siRNA was paralleled by reduction of insulin signaling through gC1qR, through the insulin receptor, and prevented adipogenesis. The rafts also were home to a variety of mitochondrial proteins during adipogenesis. Kim, K.‐B. et al., Proteomics 2009, 9, 2373‐2382. E. coli chaperone SurA is recognized SurA was a sad protein. It was sad because it couldn't get promoted without proof that it had done a good job on its current assignment. But what was that assignment? Being a good little protein, it did its best to never make a mistake and its good was very good, making thousands of perfect cycles. Still, no‐one noticed. Then one day, Vertommen et al. decided to give SurA a rest (actually its clone rested). After creating the deletion clone, they fired up the proteome machines to see what had changed. The lab was quiet as the proteomers collected their results. They sat down with the data and looked and talked, studied and talked. They finally came to a conclusion: SurA was indeed a chaperone and was responsible for transport of eight important bbarrel proteins across the periplasmic space to the outer membrane! And now a publication! Vertommen, D.. et al., Proteomics 2009, 9, 2432‐2443. Aphid saliva: solvent, glue, caulk, . . . Children learn quickly that if they don't wash their faces properly, a mother's wet thumb will finish the job. If hair won't stay where it belongs, you can always use saliva. Spots on your glasses or your computer monitor? Aphids and mosquitoes extend the uses even further. Carolan et al. report on the active components of saliva of the pea aphid (Acrythosiphon pisum), an agricultural pest that attacks legumes. The researchers used mass spectrometry, RNAi, and various types of electrophoresis to identify the nine proteins secreted in pea aphid saliva. From the complete genome sequence, four proteins could be identified by homology: a metalloprotease [M2], a zinc [M1] protease, both probably cleaving plant defensive peptides, a glucose oxidoreductase that probably detoxifies phytochemicals, and a relative of regucalsin, which might suppress Ca+2 mediated defense. Three of the proteins could not be matched to any known proteins. Carolan, J. C. et al., Proteomics 2009, 9, 2457‐2467.     

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.     

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.     

Proteomic analysis of urine in rats chronically exposed to fluoride

Urine is an ideal source of materials to search for potential disease‐related biomarkers as it is produced by the affected tissues and can be easily obtained by noninvasive methods. 2‐DE‐based proteomic approach was used to better understand the molecular mechanisms of injury induced by fluoride (F^?) and define potential biomarkers of dental fluorosis. Three groups of weanling male Wistar rats were treated with drinking water containing 0 (control), 5, or 50 ppm F^? for 60 days (n = 15/group). During the experimental period, the animals were kept individually in metabolic cages, to analyze the water and food consumption, as well as fecal and urinary F^? excretion. Urinary proteome profiles were examined using 2‐DE and Colloidal Coomassie Brilliant Blue staining. A dose‐response regarding F^? intake and excretion was detected. Quantitative intensity analysis revealed 8, 11, and 8 significantly altered proteins between control vs. 5 ppm F^?, control vs. 50 ppm F^? and 5 ppm F^? vs. 50 ppm F^? groups, respectively. Two proteins regulated by androgens (androgen‐regulated 20‐KDa protein and α‐2μ‐globulin) and one related to detoxification (aflatoxin‐B1‐aldehyde‐reductase) were identified by MALDI‐TOF‐TOF MS/MS. Thus, proteomic analysis can help to better understand the mechanisms underlying F^? toxicity, even in low doses. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:8–14 2011; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20353     

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.     

Helicobacter pylori proteomics by 2‐DE/MS, 1‐DE‐LC/MS and functional data mining

With its predicted proteome of 1550 proteins (data set Etalon) Helicobacter pylori 26695 represents a perfect model system of medium complexity for investigating basic questions in proteomics. We analyzed urea‐solubilized proteins by 2‐DE/MS (data set 2‐DE) and by 1‐DE‐LC/MS (Supprot); proteins insoluble in 9 M urea but solubilized by SDS (Pellet); proteins precipitating in the Sephadex layer at the application side of IEF (Sephadex) by 1‐DE‐LC/MS; and proteins precipitating close to the application side within the IEF gel by LC/MS (Startline). The experimental proteomics data of H. pylori comprising 567 proteins (protein coverage: 36.6%) were stored in the Proteome Database System for Microbial Research ( http://www.mpiib‐berlin.mpg.de/2D‐PAGE/ ), which gives access to raw mass spectra (MALDI‐TOF/TOF) in T2D format, as well as to text files of peak lists. For data mining the protein mapping and comparison tool PROMPT ( http://webclu.bio.wzw.tum.de/prompt/ ) was used. The percentage of proteins with transmembrane regions, relative to all proteins detected, was 0, 0.2, 0, 0.5, 3.8 and 6.3% for 2‐DE, Supprot, Startline, Sephadex, Pellet, and Etalon, respectively. 2‐DE does not separate membrane proteins because they are insoluble in 9 M urea/70 mM DTT and 2% CHAPS. SDS solubilizes a considerable portion of the urea‐insoluble proteins and makes them accessible for separation by SDS‐PAGE and LC. The 2‐DE/MS analysis with urea‐solubilized proteins and the 1‐DE‐LC/MS analysis with the urea‐insoluble protein fraction (Pellet) are complementary procedures in the pursuit of a complete proteome analysis. Access to the PROMPT‐generated diagrams in the Proteome Database allows the mining of experimental data with respect to other functional aspects.     

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.     

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.     

Cover Picture: Proteomics 9/2009

In this issue of Proteomics you will find the following highlighted articles: Rafting on the pond It seems that any river with a drop of more than 20‐30 cm/km is a candidate for a commercially viable rafting business. Biochemical rafters are pickier. They need a detergent‐resistant lipid raft where they can set up their signaling system. Kim et al. examined the changes in the raft molecules involved in insulin stimulated pre‐adipocyte to adipocyte differentiation (adipogenesis). A substantial number of adipocyte raft‐specific proteins were identified by immunoblots and confirmed by 2‐DE MS. A protein of particular interest was gC1qR, specific for mature adipocyte rafts, which also binds complement C1q and a number of other extracellular proteins (vitronectin, fibrinogen, hyaluronic acids . . .). Down‐regulation of gC1qR by siRNA was paralleled by reduction of insulin signaling through gC1qR, through the insulin receptor, and prevented adipogenesis. The rafts also were home to a variety of mitochondrial proteins during adipogenesis. Kim, K.‐B. et al., Proteomics 2009, 9, 2373‐2382. E. coli chaperone SurA is recognized SurA was a sad protein. It was sad because it couldn't get promoted without proof that it had done a good job on its current assignment. But what was that assignment? Being a good little protein, it did its best to never make a mistake and its good was very good, making thousands of perfect cycles. Still, no‐one noticed. Then one day, Vertommen et al. decided to give SurA a rest (actually its clone rested). After creating the deletion clone, they fired up the proteome machines to see what had changed. The lab was quiet as the proteomers collected their results. They sat down with the data and looked and talked, studied and talked. They finally came to a conclusion: SurA was indeed a chaperone and was responsible for transport of eight important bbarrel proteins across the periplasmic space to the outer membrane! And now a publication! Vertommen, D.. et al., Proteomics 2009, 9, 2432‐2443. Aphid saliva: solvent, glue, caulk, . . . Children learn quickly that if they don't wash their faces properly, a mother's wet thumb will finish the job. If hair won't stay where it belongs, you can always use saliva. Spots on your glasses or your computer monitor? Aphids and mosquitoes extend the uses even further. Carolan et al. report on the active components of saliva of the pea aphid (Acrythosiphon pisum), an agricultural pest that attacks legumes. The researchers used mass spectrometry, RNAi, and various types of electrophoresis to identify the nine proteins secreted in pea aphid saliva. From the complete genome sequence, four proteins could be identified by homology: a metalloprotease [M2], a zinc [M1] protease, both probably cleaving plant defensive peptides, a glucose oxidoreductase that probably detoxifies phytochemicals, and a relative of regucalsin, which might suppress Ca+2 mediated defense. Three of the proteins could not be matched to any known proteins. Carolan, J. C. et al., Proteomics 2009, 9, 2457‐2467.     

Cover Picture: Proteomics 12/2008

In this issue of Proteomics you will find the following highlighted articles: TAP tag! You're it! TAP tag is a considerably more sophisticated game than the one we played as kids. For one, the tag is something actual as opposed to that ethereal “it” which was attached to your being by unknown forces only extant during recess period. In this technical note, Kito et al. describe a clever way of using the Tandem Affinity Purification protocol coupled to stable mass isotope labeling to study the character of the association of molecules in complexes. By mixing a TAP⁺/mass isotope⁺ tagged molecule with untagged molecules before or after affinity purification, they could distinguish stable associations from transient association from spurious noise. With some additional improvements, they should be able to generate quantitative interaction information such as the off and on rates of individual components. Kito, K. et al., Proteomics 2008, 8, 2366–2370. Rafting into place: Malaria moves machinery of infection About two million people die each year of malaria. The disease is mosquito‐borne, caused by Plasmodium falciparum in humans, P. berghei in rodents. During the mammalian phase of its life cycle, the microbe multiplies in enucleated erythrocytes, regular red blood cells (RBC). The RBC is modified extensively for Plasmodium replication. Di Girolamo et al. here report their exploration of the role of “rafts” of detergent‐resistant membranes in sorting and positioning proteins essential to malarial replication. They applied proteomic techniques to membrane fractions and found rafts carried both malarial and host components. Plasmodial raft proteins were up‐ or down‐regulated by P. berghei genes at specific stages of the plasmodial life cycle. However, there also appear to be host factors that are used to internalize selected parasite products. The raft association seems to be quite dynamic for the erythrocyte phase, particularly with multifunctional protein 14–3‐3, known for regulating protein localization. Di Girolamo, F. et al., Proteomics 2008, 8, 2500–2513. A multi‐dimensional proteomic analysis of ischemia‐reperfusion injury Cardiac surgery could be said to have a temporary mortality rate (ischemic arrest) of ～100%, but the operative rate is generally <10%. A third metric is survival – roughly 24% of high‐risk patients will die within 3 years after surgery. The problem is due primarily to the effects of reperfusion at the conclusion of the surgery. Fert‐Bober et al. report here on the proteomes of rat heart proteins at various times post‐surgery, with or without reperfusion. Hearts were subjected to 0, 15, 20–25 and 30 min of post ischemia perfusion then tested for gelatinase and for mechanical function before selecting those destined for proteome determination. Samples were analyzed by 2‐DE, MALDI/TOF‐MS, and Coomassie staining. The findings were striking: most spots showed increased intensity if the hearts had not been reperfused and the converse if they had. Both sets of 2‐DE spots included metabolism enzymes, muscle components, anti‐oxidant and stress proteins. Fert‐Bober, J. et al., Proteomics 2008, 8, 2543–2555.     

In this issue: Proteomics 12/2008

In this issue of Proteomics you will find the following highlighted articles: TAP tag! You're it! TAP tag is a considerably more sophisticated game than the one we played as kids. For one, the tag is something actual as opposed to that ethereal “it” which was attached to your being by unknown forces only extant during recess period. In this technical note, Kito et al. describe a clever way of using the Tandem Affinity Purification protocol coupled to stable mass isotope labeling to study the character of the association of molecules in complexes. By mixing a TAP⁺/mass isotope⁺ tagged molecule with untagged molecules before or after affinity purification, they could distinguish stable associations from transient association from spurious noise. With some additional improvements, they should be able to generate quantitative interaction information such as the off and on rates of individual components. Kito, K. et al., Proteomics 2008, 8, 2366–2370. Rafting into place: Malaria moves machinery of infection About two million people die each year of malaria. The disease is mosquito‐borne, caused by Plasmodium falciparum in humans, P. berghei in rodents. During the mammalian phase of its life cycle, the microbe multiplies in enucleated erythrocytes, regular red blood cells (RBC). The RBC is modified extensively for Plasmodium replication. Di Girolamo et al. here report their exploration of the role of “rafts” of detergent‐resistant membranes in sorting and positioning proteins essential to malarial replication. They applied proteomic techniques to membrane fractions and found rafts carried both malarial and host components. Plasmodial raft proteins were up‐ or down‐regulated by P. berghei genes at specific stages of the plasmodial life cycle. However, there also appear to be host factors that are used to internalize selected parasite products. The raft association seems to be quite dynamic for the erythrocyte phase, particularly with multifunctional protein 14–3‐3, known for regulating protein localization. Di Girolamo, F. et al., Proteomics 2008, 8, 2500–2513. A multi‐dimensional proteomic analysis of ischemia‐reperfusion injury Cardiac surgery could be said to have a temporary mortality rate (ischemic arrest) of ～100%, but the operative rate is generally <10%. A third metric is survival – roughly 24% of high‐risk patients will die within 3 years after surgery. The problem is due primarily to the effects of reperfusion at the conclusion of the surgery. Fert‐Bober et al. report here on the proteomes of rat heart proteins at various times post‐surgery, with or without reperfusion. Hearts were subjected to 0, 15, 20–25 and 30 min of post ischemia perfusion then tested for gelatinase and for mechanical function before selecting those destined for proteome determination. Samples were analyzed by 2‐DE, MALDI/TOF‐MS, and Coomassie staining. The findings were striking: most spots showed increased intensity if the hearts had not been reperfused and the converse if they had. Both sets of 2‐DE spots included metabolism enzymes, muscle components, anti‐oxidant and stress proteins. Fert‐Bober, J. et al., Proteomics 2008, 8, 2543–2555.