In this issue: Proteomics 1/2008

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

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

In this issue: Proteomics 20/2008

In this issue of Proteomics you will find the following highlighted articles: Comprehensively confirmed annotation of short ORFs During the California gold rush, prospectors did not look only for large nuggets to pave their road to riches – they also collected gold dust. The proteomic equivalent of dust is the small ORF. It does not pay to put a lower limit on dust or ORF size because almost any size may be turned into “cash” or an important peptide. Wei et al. demonstrate how a combination of conventional genomics and proteomics can find small expressed ORFs in noisy mixtures. Their test system is Shigella flexneri rRNA genes. First, cells were lysed, pre‐fractionated through 100 kDa and 10 kDa cutoff membranes and split into two aliquots, one treated with trypsin the other not. Both were analyzed by LC‐MS/MS and queried for ORFs >15 amino acids. Total labeled RNA probed a DNA array, and, after BLAST classification, revealed 36 new small proteins. Wei, C. et al., Proteomics 2008, 8, 4209–4213. The end of an era: Late stage proteome of cheese bacterium The bacterium Streptococcus thermophilus is a common ingredient in food, serving as a starter culture for a wide variety of dairy foods, yogurt in partic­ular, in co‐culture with Lactobacillus delbrueckii ssp. bulgaricus. It is closely related to several pathogenic species but lacks critical genes for pathogenicity. Instead, it is better optimized for lactose digestion than even a teen‐aged boy. Specific genes transport lactose into the cell where it is cleaved by a β‐galactosidase. Because milk is poor in amino acids, some strains of S. thermophilius bring their own protease to digest milk peptides and proteins. This work by Herve‐Jimenez et al. finds that in the latest stage of fermentation a number of critical changes occur: carbon sources are diversified, nitrogen uptake and biosynthesis increase, and there is a dramatic up‐regulation of the entire sulfur‐containing amino acid synthetic pathways. Stimulation by L. bulgaricus is a more complex effect. Herve‐Jimenez, L. et al., Proteomics 2008, 8, 4273–4286. 2000‐spot 2‐D DIGE gels yield interesting new CJD marker from CSF Creutzfeld‐Jakob disease (CJD) is a rare, fatal neurodegenerative disease, caused by prions. It is normally seen in either a sporadic (sCJD) or variant (vCJD) form. Differential diagnosis is difficult because the few tests available are weak in sensitivity or specificity, or have not been thoroughly validated. Brechlin et al. report here a much improved cerebrospinal fluid test based on 2‐D DIGE technology and improved sample preparation. Samples were concentrated 20‐fold, then depleted of serum albumin and immunoglobulins, and, finally, labeled with CyDyes. When separated on 24‐cm pH 4 – 7 IPG strips, then on 10 % PAGE‐SDS second dimension gels, >2000 spots could be resolved. After spot analysis for significant differences (volume, p‐value, variance) five spots remained and 4 of the 5 were identified by MALDI‐TOF‐MS. The unidentified spot showed the highest discrimination (p <0.001). Identification work for the fifth spot is continuing. Brechlin, P. et al., Proteomics 2008, 8, 4357–4366.     

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.     

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.     

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.