What makes snakes attack | High-density protein ... - ACS Publications

Oct 6, 2006 - What makes snakes attack | High-density protein microarrays | A selection of new relative quantitation methods | Proteomics for vaccine ...
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What makes snakes attack

swallow it. Of the proteins that were tested, only two elicitWhat makes a frog so attractive to a hungry snake? Proteins ed an attack response in all of the trials. One of the proteins on a frog’s skin might be the cause, was 92% similar to a parvalbumin, and say Ruddy Wattiez and co-workers the other was 88% similar to b parvalat the University of Mons-Hainaut bumin. Both identifications were con(Belgium). Snakes rely on chemifirmed by western blotting. cal sensing for many behaviors, Wattiez and co-workers were surincluding prey selection. Curiously, prised to discover that parvalbumins although much is known about the were responsible for the snake-atsnake vomeronasal organ, which tack response, because these prodetects the chemical signals, very teins are known to be in frog muscle little is known about the signals cells but not on the surface of frog skin. themselves. So Wattiez and coSo to confirm that a and b parvalbuMmmm. Snakes like pasta coated with parvalbuworkers studied the frog mucus mins really could be responsible for the min. (Adapted with permis­sion. Copyright 2006 proteome to determine whether sesnake response, the researchers puAmerican Society for Biochemistry and Molecular creted proteins attract snakes. The rified these proteins from frog muscle Biology.) researchers identified two proteins by semipreparative SDS-PAGE and rethat caused snakes to attack. peated the bioassay. Again, the tests Using semipreparative SDS-PAGE, the researchers isolatwere positive. By immunohistochemistry, the researchers dised each protein from the frog mucus proteome. These procovered that cells in the dermal mucous glands of the frog teins were screened for activity in a bioassay in which a lure contain parvalbumins. Mucous glands apparently secrete of cooked macaroni was coated with one of the isolated proparvalbumins, which are then sensed by snakes, say the reteins and placed near a snake. A protein was scored as possearchers. (Mol. Cell. Proteomics 2006, doi 10.1074/mcp. itive in the assay if the snake lunged at the lure and tried to M600205-MCP200)

High-density protein microarrays Philipp Angenendt and colleagues at the German Cancer Research Center, the Max Planck Institute for Molecular Genetics, and RiNA GmbH (all in Germany) have developed a new way to make protein microarrays. The researchers use the multiple spotting technique (known as MIST) to deposit reagents, such as template DNA and a transcription/translation mix, onto a glass slide. Although it is similar to the nucleic acid programmable protein array (NAPPA) method, the new protocol allows researchers to use DNA templates that are not immobilized via special chemistries. In addition, the microarrays include ~25× more spots than NAPPA-produced microarrays. The protein microarrays are fashioned by spotting DNA templates, such as unpurified PCR products, onto coated glass slides. In a subsequent step, a cellfree transcription/translation mix is deposited on top of each DNA spot. Next, the slide is placed in a humid chamber; this incubation rehydrates the spots, allowing transcription and translation to occur. Finally, expressed proteins are © 2006 American Chemical Society

detected with antibodies. Angenendt and colleagues tested the technique by spotting plasmids containing the gene for green fluorescent protein (GFP) in increasing concentrations for various times. The signal intensity increased up to 2 h but declined slightly at the overnight time point. The researchers could detect proteins generated from as little as ~35 fg of an unpurified PCR-amplified GFP gene. Aminopropyltriethoxysilane (APTES) and Ni-chelate coatings were evaluated for the retention of translated proteins. The Ni-chelate coating resulted in spots that were slightly brighter than those on APTES-coated surfaces. The GFPs in this experiment contained 6His tags, which have been reported to bind specifically to Ni-chelate surfaces. However, in a separate experiment, the researchers tested tagged and untagged versions of GFP, and both forms adhered to the Nichelate surface to the same extent. The researchers say that APTES and Ni-chelate surfaces probably bind proteins to a slide in a nonspecific manner. Finally, the researchers PCR-amplified 384 clones from a human fetal-brain expression library and deposited the

amplicons on a Ni-chelate slide. The same samples were placed in microplate wells to compare the microarray method to a more conventional detection technique. Identical results were obtained from both experiments, so the researchers say that detection does not suffer because smaller volumes are used with the microarray technique. (Mol. Cell. Proteomics 2006, doi 10.1074/mcp. T600024-MCP200)

A selection of new relative quantitation methods Because several of the current methods for comparing protein levels among samples are expensive or complicated, many researchers are developing their own relative quantitation methods. Recently, three new approaches have been added to the mix. Arginine and histidine residues are labeled in one approach, and N-terminal amino groups are labeled in the others. Only peptides that contain arginine and histidine residues are isolated in the method developed by Luis Javier Gon­ zález and co-workers at the Center for Genetic Engineering and Biotechnology and the National Center for Scientific

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currents Wrestling with SUMOs Some posttranslational modifications (PTMs) can be identified by a neutral loss in MS/MS experiments. But it can be ­nearly impossible to identify others, such as small ubiquitin-like modifiers (SUMOs), that fragment in an MS/ MS run. The peaks produced by the fragmenta­tion of these PTMs can overlap with those due to the target peptide. Brian Raught and co-workers at the Institute for Systems Biology, the Ontario Cancer Institute and McLaughlin Centre for Molecular Medicine (Canada), Johns Hopkins University, the Swiss ­F ederal Institute of Technology, and the University of Zurich, therefore, have ­developed SUMmOn, a pattern-recognition program that automatically teases out SUMO peaks from complex spectra. The researchers say that although the program originally was designed to detect SUMOs, it also can be applied to any modification that produces diagnostic fragment ions. Raught and co-workers modified a fragment of RanGAP1 with SUMO-1, ‑2, and -3, treated the reaction mix with trypsin, and analyzed the peptides by MS/MS. Sequest and Mascot did not identify SUMOylated peptides, but SUMmOn did. The new program also detected SUMO multimers. In addition, the researchers determined the locations of multimeric SUMO-1 attachments on a fragment of Nup358. (Nat. Methods 2006, 3, 533–539)

Unique protein sequence identifiers Several protein-sequence databases exist, and each of them uses its own accession numbers, or identifiers. To get everyone on the same page, Carol Gio­ metti and György Babnigg at Argonne National Laboratory have generated a database of unique IDs called sequence globally unique identifiers (SEGUIDs). SEGUIDs are stable and are derived from the primary sequences of proteins. Annotations and alias identifiers obtained from other databases are linked to SEGUIDs and can be visualized in a table. The database, which contains data for >2.5 million sequences, can be downloaded at http://bioinformatics. anl.gov/SEGUID. (Proteomics 2006, 6, 4514–4522)

Research (both in Cuba). To compare two protein samples, the researchers digested equal amounts of each with lysylendopeptidase and trypsin. Peptides are blocked at their a- and -amino groups with deuterated or nondeuterated acetic anhydride; this process in­troduces tags for relative quantitation and restricts the positive charges to the arginine and histidine residues. The samples are mixed, desalted, and then run on a strong-cation-exchange column. Neutral peptides are discarded, and multiply charged peptides are analyzed by LC/MS/MS. The researchers apply the method to a model protein and several protein mixtures. According to the researchers, ~84% of proteomes can be quantitated with the technique. One drawback is that tyrosines also can be modified; this side reaction could complicate analyses. (Proteomics 2006, 6, 4444–4455) Mark Knepper and co-workers at the National Heart, Lung, and Blood Institute have developed the in vacuo isotope-coded alkylation technique (IVICAT) in which the N-terminal amino groups of peptides are labeled. Peptides are lyophilized, then incubated with deuterated or nondeuterated methyl iodide overnight in a vacuum. Methyl esters are removed, and the methylated peptides are evaporated and resus-

Proteomics for vaccine development

pended in MS-compatible buffer. The trimethylammonium group adds a permanent positive charge to the N-termini and increases the signal intensities of the peptides in LC/MS/MS runs. Various peptides and proteins were labeled and compared with the IVICAT method. The researchers observed an anomalous side reaction when angiotensin II was labeled; they say that the reason for the deamination of the N-terminal aspartic acid residue is not clear. (Rapid Commun. Mass Spectrom. 2006, 20, 2463–2477) Metal element chelated tags are used by Xiaohong Qian and co-workers at the Beijing Institute of Radiation Medicine, the Beijing Proteome Research Center, and the Beijing Institute of Transfusion Medicine. DiethylenetriamineN,N,N´,N˝,N˝-pentaacetic acid (DTPA) is attached to the N-terminal amino group of peptides. DPTA is chelated to the rare earth metals yttrium or terbium. An internal lysine in some peptides also was labeled. The process was rapid; DPTA attachment took ~1 h, and tagging with metals was nearly instantaneous. Several peptides and proteins were analyzed with the method. An ion suppression effect was observed, but multiplying the experimentally obtained ratio by a rectifying coefficient compensated for it. (Anal. Chem. 2006, 78, 6614–6621)

83 proteins from the >100 spots. A total of eight proYou’ve planned for your vateins were selected for vaccation abroad for months. cine studies. These proBut instead of seeing the teins were chosen because sights, you spend the first they had a low similarity to day of your holiday in the proteins in humans and E. restroom. You have a case coli and were not studied of “traveler’s diarrhea”. To previously as vaccine canhelp travelers avoid this didates. The proteins were A clinical use. miserable outcome, P. expressed in E. coli and Proteomics can help Schrotz-King and colpurified. Mice were immuresearchers develop leagues at ACE BioSciencnized with the proteins at vaccines to protect against infections. es A/S (Den­mark) plan to 3 time points and analyzed develop a vaccine against for resistance to C. jejuni. Campylobacter je­juni, the For 2 proteins (ACE961 and main organism re­sponsible for the ACE1569), the number of bacterial cells condition. As a first step, they used in the feces of the mice was greatly rea proteomics approach to identify C. duced relative to a control inoculation. jejuni antigens that could be used in The researchers say that protein-based a ­vaccine. vaccines, therefore, could be useful for Polypeptides were isolated from the preventing traveler’s diarrhea and other surfaces of C. jejuni cells and separatconditions. (Vaccine 2006, doi 10.1016/ ed by 2DE. The researchers identified j.vaccine.2006.05.085)

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PHOTODISC

Toolbox

Fission yeast “localizome”

Nucleolus

Nuclear envelope

MINORU YOSHIDA

currents

Knowing where proteins are Prh1 Cut15 within a cell can give you information about their functions. So Minoru Yoshida and co-workers at RIKEN, the Japan Science and Technology Agency, the University of Tokyo, and the National Institute of Information and Communications Technology Microtubule Periphery (all in Japan) have determined the locations (the “localizome”) Spac24b11.07c Ght2 of most of the proteins expressed in the fission yeast Schizosaccharomyces pombe. By comparing the localizome before and after they treated cells with an inhibitor, the researchers also discovered that the export of several proteins from the nucleus deVacuole membrane Endoplasmic reticulum pends on a protein called Crm1. Gpi16 Spac2f7.10 To test the protein expression from S. pombe genes, the researchers placed 4910 open reading frames (ORFs) into vectors that included a His tag sequence; these plasmids represent 99.2% of the predicted ORFeome. The ORFs were integrated into the genome with an inducible proNucleus Mitochondrion moter, so the His-tagged proteins Rsm10 Alp13 were expressed when thiamine was absent from the media. After induction, cell lysates from each strain were spotted onto nitrocellulose membranes, and this macroarray was immunoblotted with an anti-His antibody. Protein levels varied widely, despite Where are you? Representative localizations of some the fact that all of the ORFs had of the 4431 S. pombe proteins analyzed in a global the same regulatory elements. study of the “localizome”. mRNA levels were more consistent and did not correlate with protein levels. the localizome data could be used to deFor subcellular localization studies, termine protein functions. They treated the ORFs were cloned into vectors that S. pombe cells with leptomycin B, an contained the sequence for yellow fluoinhibitor of Crm1, which is an exportin rescent protein. Again, the ORFs were that moves other proteins from the nuintegrated into the genome with the cleus into the cytoplasm. Data obtained same inducible promoter. All 4948 ORFs after treatment were compared with the of S. pombe were analyzed in this part original localizome. The positions of 285 of the investigation. Fluorescent signals proteins, including the 5 known Crm1were observed for proteins expressed dependent proteins, were identified. The from 4431 ORFs. When Yoshida and coresearchers say that although this protoworkers classified the localizations into col results in higher than normal levels categories, they found that the distribuof protein expression that could cause tion of proteins for S. pombe was similar artifacts, it still provides useful data that to that reported previously for another would be very difficult to obtain with yeast, Saccharomyces cerevisiae. less global methods. (Nat. Biotechnol. The researchers demonstrated that 2006, 24, 841–847)

Toolbox QUIL for quantitation Rong-Fong Shen and co-workers at the National Heart, Lung, and Blood Institute have developed an easy-to-load algorithm called quantitation using isotope labeling (QUIL) that automatically determines the relative amounts of proteins in multiple samples. After a list of identified proteins and peptides is generated, researchers apply QUIL to the MS n data for quantitation. For each set of labeled and unlabeled peptides, QUIL produces an ion chromatogram with the background noise subtracted. To determine the beginning and end of an ion peak, QUIL uses an approach based on the full width at half-maximum value; this method is tolerant to irregularly shaped peaks. The areas and ratios of the labeled and unlabeled pairs are calculated, and statistical tests are performed with the program. The researchers applied QUIL to the study of a standard protein and two complex protein mixtures labeled at various ratios with 16 O/ 18 O or the isotope-coded affinity tag (known as ICAT) method. (Anal. Chem. 2006, 78, 5752–5761)

PEPPeR Steven Carr and colleagues at the Broad Institute and the Harvard Medical School have developed algorithms that allow researchers to combine pattern-matching techniques with identification techniques for biomarker discovery. Together, the algorithms comprise the platform for experimental proteomic pattern recognition (PEPPeR). With the landmark-matching algorithm, researchers use relative chromatographic elution data to align peaks from a subset of peptides in a complex mixture with peaks that have been identified in previous experiments. The ­order of the landmark peptides helps researchers to compare data sets obtained from two conditions, such as healthy and diseased. The peak-matching algorithm consistently recognizes known and novel peaks across multiple experiments for label-free quantitation. The patterns of the peak intensities are analyzed for differential expression between samples. Peaks from unknown peptides can be identified by MS/MS. (Mol. Cell. Proteomics 2006, doi 10.1074/mcp.M600222-MCP200)

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