currents
Zebrafish proteomics
additional wash steps are performed. The zebrafish Danio rerio has become Samples can be frozen or used immean important model organism for the diately in gel analyses. study of embryogenesis. Because zeAlthough this method decreased brafish are transparent, researchers can the total amount of protein per emEmbryo easily visualize the ballet of cell movebryo, the amount of cellular protein ments and divisions that occur as the per embryo remained nearly conorganism takes shape. However, a mastant. Removing the yolk allowed the jor challenge for proteomics scientists researchers to load protein from sevwho study zebrafish embryogenesis eral more embryos onto gels. By anhas been the presence of highly abunalyzing Coomassie-stained 1DE gels, Yolk cell dant yolk proteins that obscure the lessthe researchers determined that the plentiful embryo proteins on 2DE gels. levels of yolk proteins were greatly reSo Carl-Philipp Heisenberg and colduced when the deyolking procedure leagues at the Max Planck Institute of was performed. Whereas embryo Molecular Cell Biology and Genetics protein signals had been swamped (Germany) have developed a protocol by yolk protein signals when convento remove yolks from multiple embryos tional sample-preparation methods simultaneously. The resulting embryo were used, many cellular proteins beBringing up baby. An early-stage zebrafish sample is compatible with subsequent came visible on 2DE gels in the abembryo. (Adapted with permission. Copyright 1DE and 2DE analyses. sence of yolk proteins. 2006 Link et al.; licensee BioMed Central Ltd.) In the new method, Heisenberg and The researchers also analyzed 57 colleagues use mechanical stress to protein spots by MALDI TOFMS and disrupt yolk cells. Because yolk cells are less stable than emcompared the identification rates of three zebrafish sequence bryos, simply pipetting an embryo with a narrow tip tears the databases. A total of 51 proteins were identified. According to yolk cell. Up to 100 embryos can be handled at once. The the researchers, >95% of the identifications were made ussample is shaken at 1100 rpm for 5 min and pelleted to reing a combination of two databases: Ensembl and MS Datamove the yolk contents. The supernatant is removed, and two Base. (BMC Dev. Biol. 2006, 6, 1)
Forever young? We’re all searching for the fountain of youth. Products purported to restore youth and vitality compose a booming industry. But what really works? Kwang Hoon Lee and co-workers at Yonsei University College of Medicine (Korea) tested four potential anti-aging agents on endothelial cells and used various assays and proteomics methods to evaluate the results. Although epithelial cells are typically used for aging studies, Lee and co-workers chose endothelial cells because these cells are exposed to both blood and tissue and are involved in homeostasis in vivo. Endothelial cells line blood vessels and regulate the exchange of molecules, gases, and liquids. In addition, several vascular diseases, such as atherosclerosis, hypertension, and congestive heart failure, are related to aging. To obtain “old” cells, the researchers harvested human dermal microvascular endothelial cells after 20–25 passag© 2006 American Chemical Society
and few of the treated cells were positive. Only kinetin and selenium affected cell viability and proliferation, and only kinetin-treated cells had a significantly i ncreased cell metabolism. Lee and co-workers also compared 2DE gels of proteins from young, un(a) (b) (c) treated old, and treated cells. Moesin, rho guanosine-5′-diphosphate-dissoEarly passage ciation inhibitor (rho GDI), and actin were up-regulated in young cells and Late passage treated cells. In western blot analyses, however, only moesin levels were Youthful appearances. (a) Young cells and (c) kinetinsignificantly high in all of the treated treated old cells closely resemble each other, but both cells and in young cells. Rho GDI levlook different from (b) untreated old cells. (Scale bar = els were high only in kinetin- and se20 µm) (Adapted with permission. Copyright 2006 WileyVCH Verlag GmbH & Co.) lenium-treated cells, and actin did not appear to be differentially expressed among the cell lines. The levcells and with “young” cells harvested els of several cell-cycle proteins were deafter 10 passages. creased in treated cells and in young Morphologically, treated cells closely cells. The researchers say that all of the resembled young cells. Whereas most of agents delayed aging, but only kinetin the untreated old cells were positive for additionally increased cell proliferation aging in a senescence-associated b-gaand metabolism. (Proteomics 2006, 6, lactosidase assay, none of the young cells 1351–1361) es. In various tests, old cells that had been treated with the potential anti-aging agents kinetin, epigallocatechin-3gallate, all-trans retinoic acid, and selenium were compared with untreated old
Journal of Proteome Research • Vol. 5, No. 4, 2006 735
currents Toolbox QualScore Few of the hundreds of thousands of MS/MS spectra generated in a shotgun proteomics experiment are confidently assigned to a particular peptide sequence. Typically, those spectra that are not assigned are disregarded. But according to Alexey Nesvizhskii and co‑workers at the Institute for Systems Biology, the Swiss Federal Institute of Technology, and the University of Zu rich, such spectra may be unassigned at this step simply because posttranslational or chemical modifications or single-nucleotide polymorphisms are present. To salvage useful data from these spectra, these researchers have developed a tool called QualScore. QualScore learns the characteristics of high-quality spectra from the first pass of the data through an MS/MS search engine. The tool automatically develops a statistical classifier for each dataset on the basis of this information; this step obviates the need to rely on training data sets. The classifier is then applied to the unassigned spectra to identify high-quality spectra that were previously overlooked. When QualScore was used on a human lipid-raft data set, the tool increased the number of identified high-quality spectra by 20%. QualScore is freely available at www. proteomecenter.org. (Mol. Cell. Proteo mics 2006, doi M500319-MCP200)
Minimotif Miner Motifs are short, conserved protein sequences that can serve as sites for post translational modifications and protein– protein interactions. In addition, motifs can function as signals for protein trafficking. Martin Schiller and co-workers at the University of Connecticut and the University of Connecticut Health Center have developed a web-based motif search tool called Minimotif Miner (MnM) and a motif database to enable researchers to locate short-sequence motifs in proteins. Whereas most motif databases contain only a subset of motifs, the new database contains a comprehensive group. The MnM tool can identify motifs and rank them on the basis of three scoring functions to reduce false positives. MnM is available at http://mnm.engr.uconn.edu. (Nat. Methods 2006, 3, 175–177)
Genome-wide screen for complexes Protein complexes drive many cellular processes, such as transcription. To identify complexes and learn how these protein machines work, Giulio SupertiFurga, Robert Russell, and colleagues at Cellzome AG, the European Molecular Biology Laboratory, the Max Planck Institute for Molecular Genetics, the Max Planck Institute for Infection Biology (all in Germany), and the Center for Molecular Medicine of the Austrian Academy of Sciences performed the first genomewide screen for protein complexes in the yeast Saccharomyces cerevisiae. They purified 491 complexes, including 257 that are novel. The researchers used the tandem-affinity-purification method and MS to isolate proteins. Of the 6466 open reading frames that were tagged, 1993 unique fusion proteins encoded by these known and putative genes were isolated. Most of the proteins bound at least one partner. In total, 2760 distinct proteins, which represent ~60% of the yeast pro-
Proteases as biomarkers
Studies that propose the use of peptide peak patterns, or barcodes, to identify disease biomarkers have come under fire recently. Critics charge that the patterns are not reproducible and that the proteins from which the peptides are derived should be identified before clinicians implement the method. In an attempt to address some of these concerns, Paul Tempst and co-workers at Memorial Sloan-Kettering Cancer Center used an automated method to analyze peptides from cancer patients and healthy controls. According to the researchers, the discriminating peptide patterns are actually the byproducts of the real cancer biomarkers—proteases. Human serum samples from people with breast, bladder, or prostate cancer and from healthy controls were incubated with magnetic reversed-phase beads. The peptides captured by the beads were then analyzed by MALDI TOFMS. An algorithm developed by the researchers helped them to align the spectra and perform statistical tests. Reproducibility was checked by monitoring the spectra from 12 reference samples.
736 Journal of Proteome Research • Vol. 5, No. 4, 2006
teome, were identified with the method. The researchers say that because many complexes were repeatedly isolated, the screen probably was performed to saturation. The scientists developed a socioa ffinity index on the basis of their experimental data to computationally determine the likelihood that any protein would bind to other proteins. They compared the socio-affinity index scores with known dissociation constants and data obtained from 3D structures and two-hybrid assays for some of the protein pairs. The scores correlated well with these known data; this finding suggests that the scores are functionally relevant. The scores were also used to define complexes containing more than two proteins. The computational analysis revealed that proteins in complexes are either core proteins or proteins that occasionally form attachments to cores. The researchers say that because most core components are expressed at the same time in the cell cycle and are localized
Tempst and co-workers could discriminate between diseased and control samples and among the different cancer samples by comparing the normalized intensities of 651 peaks. To make the protocol more feasible for clinical tests, the researchers reduced the number of key features to 68 with no loss of discriminating ability, by applying statistical measures. Of the 68 peptides, 46 were identified by MS/MS. Most of the diagnostic peptides clustered into groups of overlapping fragments from the same larger “founder” peptides. When tested on an independent set of prostate-cancer samples, the peptide ion signatures differentiated the cancer samples from the controls. The diagnostic peptides had specif ic cleavage patterns, and the discriminating peptide pattern did not seem to correlate with the concentrations of the founder peptides. Therefore, the re searchers concluded that cancer-specific proteases in samples from cancer patients were degrading the founder peptides after sample collection. The researchers say that they must still identify the protease biomarkers. (J. Clin. Invest. 2006, 116, 271–284)
currents to the same cellular compartments, the complexes isolated in the study are likely to be functional units. Modules, which are groups of two or more proteins that were usually isolated together, often bound to cores within the same functional classes. (Nature 2006, doi 10.1038/ nature04532)
Biomarkers of toxicity Paralytic shellfish poisoning due to toxic dinoflagellates is a serious problem, but current methods to screen water and sediment samples are expensive, timeconsuming, and ill-suited for field use. Thus, David Dudgeon and co-workers at Nankai University, the University of Hong Kong, the City University of Hong Kong, the University of Science and Technology, and Xiamen University (all in China) performed proteomics anal-
Which proteins are in your Renaissance painting?
yses of toxic and nontoxic Alexandrium tamarense strains to identify biomarkers of toxicity. Using 2DE, the researchers noted three proteins in toxic strains that did not occur in nontoxic strains. Further analysis by MALDI MS and peptide sequencing indicated that these polypeptides were isoforms of a toxin-related protein from A. minutum. The researchers also raised murine monoclonal antibodies against one of the proteins. When tested with western blot analysis, the antibodies showed no cross-reaction with other protein species and clearly distinguished between the toxic and nontoxic strains. The researchers suggest that these findings offer potential for the development of immunoassays to be used in routine monitoring. (Proteomics 2006, 6, 654–666)
efficient extraction procedure involved finely grinding the paint sample with a Art historians and restorers need to commercial synthetic resin in a 1% triknow the composition of paints for the fluoroacetic acid solution with a mordocumentation and preservation of tar and pestle, followed by a series of historical works. incubations in an But the current ultrasonic bath. analytical methThen, they studods provide litied the protein tle quantitative extraction efficiency by MALDI or structural information about TOFMS. To prevarious binding cisely identif y agents, such as the binder promilk, eggs, or teins, Cren-Olivé glue, in paints and colleagues that were used digested the exduring the Ren tracted proteins aissance. So, with trypsin and Cécile Cren-Olivé analyzed them by and colleagues LC/MS/MS. Egg-bound. A new protein extraction at the UniverThe investigamethod determined that Benedetto Bonfigli’s sity of Science tors applied their triptych The Virgin and Child, St. John the and Technology protein extracBaptist, St. Sebastian contained both egg whites and yolks as the paint binders. of Lille (France) tion and analysis and the Centre method to ~10de Recherche et µg paint samples from two paintings, Benedetto Bonfig de Restauration des Musées de France developed and tested a proteomicsli’s 15th-century triptych The Virgin and based method to identify proteins in Child, St. John the Baptist, St. Sebas paints. tian and Niccolò di Pietro Gerini’s 14thCren-Olivé and colleagues created century painting The Virgin and Child. formulations of paints to develop their Using this new proteomics method, they determined for the first time that protein extraction methods. They used both Renaissance paintings contained lead white as the pigment and either whole egg or a mixture of ovalbumin whole eggs as paint-binding agents. and linseed oil as binders. The most (Anal. Chem. 2006, 78, 1494–1502)
Toolbox Human plasma proteome analysis Jong Shin Yoo and colleagues at the Korea Basic Science Institute and Yonsei University (Korea) have developed a filtering method for the high-throughput analysis of large datasets, such as the human proteome. The new method is based on the analysis of a bacterial proteome, which is much simpler and has fewer modifications than the human proteome. Initially, the researchers analyzed the proteome of the bacterium Pseudomonas putida KT2440 and applied the so-called decoy approach, which uses a reversed-sequence database, to the PeptideProphet score. They then used the molecular weight of the protein (determined by its mobility in 1DE gels) to refine the identification result. The same protocol was then applied to the human plasma proteome and used to determine the criteria for clustering it into three groups. The approach could lead to higher-confidence protein identifications in MS/MS database searches without the need for extensive computation. (Proteomics 2006, 6, 1121–1132)
Peptide-centric database for searches Current MS/MS search programs do not always distinguish between correct and incorrect peptide sequence assignments. This distraction worsens when larger databases are used. To create smaller, more-focused databases, Katheryn Resing and colleagues at the University of Colorado at Denver have developed a method for removing unlikely sequences. In the new approach, userspecified rules are used to exclude unlikely missed cleavages and nontryptic proteolysis products. The rules were determined by data-mining >11,000 high-confidence peptide assignments. Ion-exchange chromatographic behavior was also used as an editing criterion. The researchers show that the resulting peptide-centric databases can be used in place of protein databases. The smaller databases yield better discrimination between correct and incorrect sequence assignments and seem to contain all of the critical information contained in a full protein database. (Anal. Chem. 2006, 78, 1071–1084)
Journal of Proteome Research • Vol. 5, No. 4, 2006 737
