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Self-assembling protein arrays
(GST) tag and then captured and held onto the slide by a GST antibody that was spotted next to the cDNA. In every Although protein microarrays are potentially useful for highspot, ~10 fmol of protein were produced and immobilized, an throughput screens of protein interactions, they have not amount comparable to that used in other array methods. been widely adopted. Current methods of fabricating protein To test the NAPPA method, the researchers bound microarrays involve purifying mammalian proteins, often from eight different target proteins onto the array. In separate yeast or bacteria, then spotting the proteins onto a slide. But experiments, two query proteins tagged with the hemagglupurifying mammalian proteins is difficult, and some proteins tinin (HA) epitope were assayed for interaction with the are not stable for long periods of time on an array. Joshua eight targets. Interactions were detected by probing the LaBaer and colleagues at Harvard Medical School have array for the HA tag. Each query protein interacted specifideveloped a new way of producing protein arrays that overcally with its known interacting proteins on the array. In a comes these limitations. larger experiment, the researchers identified previously Instead of purifying mammalian proteins from yeast or known and unknown bacteria, LaBaer and interactions among 29 colleagues make proTarget Target protein different proteins teins directly on a miGST antibody DNA involved in human croarray slide. ComAvidin DNA replication. plementary DNAs A few technical (cDNAs) encoding for Add cell-free expression system hurdles must still be various mammalian overcome for the proteins are adhered Stuck on you. A schematic of the NAPPA approach to immobilizing proteins onto slides for NAPPA method to onto a glass slide via protein arrays. (Adapted with permission. Copyright 2004 American Association for the detect certain binding biotin/avidin linkages Advancement of Science.) events. For example, at different spots. the presence of a When the researchthird protein may be necessary to bridge an interaction. ers are ready to use the nucleic acid programmable protein Appropriate posttranslational modifications, which may be array (NAPPA), a commonly used cell-free mammalian necessary for particular interactions, may not be present lysate is added to the entire slide surface. The lysate tranafter the proteins are translated in the NAPPA method. scribes the cDNA into mRNA and translates the mRNA into (Science 2004, 305, 86–90) protein. Each protein is fused to a glutathione-S-transferase
Assay for interactions requiring PTMs Posttranslational modifications (PTMs) affect many protein interactions. For example, some proteins must be phosphorylated or acetylated before they can bind to their partners. PTMs also can inhibit certain protein interactions. In traditional two-hybrid systems, researchers can assay only unmodified proteins. To investigate interactions that require PTMs, Min-Hao Kuo and colleagues at Michigan State University and the University of Washington adapted the twohybrid system using a tethered catalysis approach. In the new version of the assay, the protein of interest is fused to the enzyme that modifies it, and this fusion protein is used as bait. Kuo and colleagues initially demonstrated the method by expressing a fusion of a histone acetyltransferase to a histone protein. When they used it as bait in two-hybrid screens, they identified nine interacting proteins. They also performed screens with a kinase –RNA © 2004 American Chemical Society
polymerase fusion. In that case, several proteins specifically interacted with the phosphorylated polymerase. (Nat. Biotechnol. 2004, doi 10.1038/nbt985)
Brain peptidomics Robert Kennedy and colleagues at the University of Florida and the University of Michigan, Ann Arbor, have developed a technique to study neuropeptides produced in the brains of live rats. Using the new technique, the investigators identified a novel group of non-opioid peptides derived from proenkephalin A and other peptides that are biomarkers for brain trauma and synaptic activity. Kennedy and colleagues collected extracellular fluid from 13 male rats by inserting a microdialysis probe into their brains. Samples were taken both under basal conditions and after localized depolarization with a highly concentrated K+ solution, which made the cells release neurotransmitters. The samples were analyzed by capillary LC/MS/MS. The
spectra from basal and depolarization conditions were subtracted from each other, and the resultant spectra were scanned by Sequest and Mascot database searches. Spectra identified by both searches were then submitted to Lutefisk for de novo sequencing. The investigators identified 29 peptides, of which 25 were novel. The proteins included neuropeptide precursors, blood proteins, and transporters. Six non-opioid peptides were tested for neurochemical activity. The investigators delivered each of the peptides into rat brains and monitored the release of amino acid neurotransmitters. Changes in amino acid concentrations confirmed that three of the six peptides had bona fide neurochemical activity. Kennedy and colleagues suggest that their experimental method could have applications in developing drugs, treating brain trauma, and studying different physiological states, such as sleep and hunger. (Anal. Chem. 2004, 76, 5523–5533)
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TOOLbox
rat
Proteomics researchers often label cultured cells, unicellular organUnlabeled 15N-labeled Unlabeled 15N-labeled cycloheximideisms, and more control treated complex model Homogenize Homogenize organisms, such as Mix Mix the roundworm Proteolytic digestion Proteolytic digestion Caenorhabditis elegans and the fruitMudPIT MudPIT Identify peptide Measure ratio B Identify peptide Measure ratio A fly Drosophila melanogaster, by exposing them to isotopes, which bem/z Time m/z Time come incorporated in the cell or the Ratio A = Cycloheximide-treated organism. But John Control Ratio B Yates and colleagues at the A ratio of ratios. A schematic of a quantitative proteomics experiment with Scripps Research a cycloheximide-treated rat, a control rat, and an 15N-labeled rat. Institute, the University of Colorado Health Sciences Center, and the University counterpart in a ratio. Taking the ratio of of Vermont have gone a step further. They these two ratios for each peptide revealed have metabolically labeled a mammal, the changes in protein levels in response to rat Rattus norvegicus, by adding 15N-labelcycloheximide. Most of the proteins that changed in ed algae to rat food that lacked protein. All the cycloheximide-treated rat livers were of the proteins ingested by the rat, theremembrane-associated. The levels of profore, were labeled with 15N. The rats that teins involved in metabolizing xenobiotics ate the 15N-labeled food appeared to be increased with cycloheximide exposure. healthy, and they had the same growth This was expected because the liver rids curve as normal rats. the body of foreign substances. The levels Yates and colleagues used tissues from of some chaperones that are involved in a rat that ate 15N-labeled food as an interthe quality control of protein folding also nal standard in a quantitative proteomics increased. Some proteins may not have experiment. They fed the rat with the labeen fully synthesized when the rat was beled food for 44 days, then spiked liver treated with cycloheximide, and partially tissue from the labeled rat into both contranslated proteins may not fold properly. trol and cycloheximide-treated rat liver This may have stimulated an increase in samples. They digested the proteins and chaperone production or a decrease in then analyzed the peptides by LC/MS/MS. chaperone degradation. In other methods, only certain amino To validate the labeling method, three acids are labeled, whereas here, 15N is inquality-control proteins were further studcorporated into nearly all the amino acids ied by quantitative Western-blot analysis. in every protein. The amount of each unThe results were similar to those obtained labeled peptide from both the control rat by metabolic labeling. (Anal. Chem. 2004, and the cycloheximide-treated rat was 76, 4951–4959) compared separately to its 15N-labeled
Singling out proteins in mixtures What is effective for one protein can be poor for another, conclude Seetharaman Vaidyanathan and colleagues at the University of Manchester Institute of Science and Technology (U.K.) in their study of the effects of ESI-MS instrumental settings on the detection of proteins in mixtures. The researchers varied 14 instrumental parameters, including flow rates, temperatures, and voltages.
The changes influenced the detection of the proteins in a five-component mixture in very different ways. In some cases, the settings were optimal for one protein but not for the others. The settings made such a big difference that it was possible to detect individual proteins in the mixture without a separation step. The results could have implications for top-down proteomics strategies. (Anal. Chem. 2004, 76, 5024–5032)
Help for ambiguous peptide matches Sometimes, it can be difficult to make a confident match between a mass spectrum and a peptide with current search engines, such as Sequest and Mascot. In those cases, researchers typically resort to manual curation, which dramatically slows down high-throughput proteomics experiments. To speed up the identification of such peptides, Frederick Roth and colleagues at Harvard Medical School have developed a new software tool called Spectrum Intensity Likelihood Viewer (SILVER). SILVER is a Web-based program in which users upload the original tandem mass spectrum and a list of candidates generated by Sequest or Mascot. SILVER assesses the likelihood that each candidate is the correct peptide based on decision trees that were trained on >27,000 spectra. A visual display of the assessment is available within 2 s. The code is free to academic researchers who request it. SILVER can be found at http:// llama.med.harvard.edu/Software. html. (J. Am. Soc. Mass Spectrom. 2004, 15, 910–912)
Database of FTIR reference spectra Although FTIR spectroscopy is a promising tool for rapidly characterizing protein secondary structure, most studies thus far have relied on a limited number of reference spectra. As a first step toward building a reliable, pattern-recognition-based system for proteomics, Parvez Haris and colleagues at De Montfort University (U.K.) have begun developing a comprehensive database of FTIR spectra for proteins of known structure. Called the protein infrared spectra databank (PISD), the database contains FTIR spectra from several different laboratories. Surprisingly, the researchers discovered that combining FTIR spectra from multiple laboratories in the PISD did not greatly affect prediction accuracy. Eventually, they hope to add FTIR spectra for all proteins with known structure to the PISD. (Proteomics 2004, 4, 2310–2319)
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TOOLbox New programs for comparative proteomics Gel-free, multidimensional shotgun LC/MS proteomics techniques are fast and sensitive, but sometimes the results from these studies are not easily reproduced. A method to determine the accurate mass and elution time of peptides exists, but it is currently only applicable to expensive FT-MS instruments. Dragan Radulovic, Andrew Emili, and colleagues at four universities and a research institute in the United States and Canada have developed a collection of algorithms, statistical methods, and other applications to evaluate large-scale protein expression profiling of mammalian samples by LC/ MS. The new programs automatically detect and quantify peptide peaks, compensate for fluctuations in signal intensities and retention times, and accurately compare peptides from different datasets. (Mol. Cell. Proteomics 2004, doi 10.1074/mcp. M400061-MCP200)
Matching high-resolution mass spectra To identify a protein in a complex mixture, researchers typically search a large database of known mass spectra for a match based on spectral similarities. Currently, all search methods require certain variables, such as the ion masses, to be identical from spectrum to spectrum. But aligning high-resolution mass spectra is not a simple task, because several narrow peaks can reside within a small area of the spectrum. To overcome this limitation, Michael Hansen and Jørn Smedsgaard at the Technical University of Denmark have developed a new algorithm that matches complex, highresolution mass spectra without relying on alignments. The researchers tested the algorithm with crude fungal extracts analyzed on a quadrupoleTOFMS instrument. The distances between the peaks in an unknown protein’s mass spectrum were compared with those in known spectra. The new program performed as well as existing methods. (J. Am. Soc. Mass Spectrom. 2004, 15, 1173–1180) 914
NMR of proteins in bilayer nanotube arrays Gary Lorigan and colleagues at Miami University have carried out the first solid-state NMR analyses on an integral membrane protein in aligned phospholipid bilayer nanotube arrays. The approach offers several advantages over using magnetically or mechanically aligned phospholipid bilayers. Aligned membrane systems provide more structural information than disoriented phospholipid bilayers, and they can be Bilayer nanotube array. (a) A substrate made of anodic aluminum used to better understand oxide contains nanopores that can be lined with oriented phosphothe dynamic properties lipid bilayers. (b) A close-up of an oriented bilayer inside a pore. of integral membrane proteins. be used to study parallel bilayers on glass The researchers created the aligned plates and perpendicular bilayers in an phospholipid bilayer nanotube arrays using array format, data from both orientations nanoporous anodic aluminum oxide (AAO) can be directly compared. Second, phosas a substrate. Within a given pore of the pholipid bilayers in an array format are AAO substrate, the phospholipids formed more rigid than lipid bilayer vesicles. This an aligned bilayer, with water in the middle. makes it easier to study low-abundance A peptide containing the transmemnuclei like 13C and 15N. Third, the pores in brane region of an integral membrane acetylenase, CREP-1, was introduced into the the AAO substrate strictly define the paraphospholipid bilayers. The protein was meters of the aligned phospholipid bilayers. found to have a helical tilt of 6° with respect The investigators suggest that analyses of to the bilayer normal. the diameter of the substrate pore and the The phospholipid bilayer nanotube length of the lipid acyl chain could be interarrays have several advantages. First, beesting. (J. Am. Chem. Soc. 2004, 126, cause the same solid-state NMR probe can 9504–9505)
Environment affects proteomes Jan Schnitzer and colleagues at the Sidney Kimmel Cancer Center and the Scripps Research Institute have mapped the proteome on the surface of endothelial cells, both in vivo and in vitro. The investigators discovered that cells can express distinctly different proteins, depending on whether they were in vivo or cultured in petri dishes. The results indicate that the microenvironment of cells can influence protein expression. The investigators studied rat-lung endothelial cells. Both in vivo and in vitro samples were subjected to >30 multidimensional protein identification technology (MudPIT) analyses. Only 51% of the proteins associated with the integral and plasma membranes were common to both in vivo and in vitro cells. This implies that the environment created by standard cell-culture procedures does not properly replicate in vivo conditions. The investigators suggest that cell-surface proteome mapping could help scientists understand not only protein expression in different environments but also changes in protein expression that result from disease states. The differences in proteomes between healthy and diseased cells may lead to the development of tissuespecific drugs and targeted gene therapies. (Nat. Biotech. 2004, 22, 985–992)
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currents Oligosaccharides labeled with cyanine dyes Akihiko Kameyama and colleagues at the National Institute of Advanced Industrial Science and Technology and Amersham Biosciences (both in Japan) have used hydrazide derivatives of cyanine dyes to label oligosaccharides for detection by MALDI-TOFMS. Cyanine reagents have a positive charge and hydrophobic properties, both of which improve the sensitivity of MALDI-TOFMS for the analysis of glycosylated proteins. The researchers detected most of the known N-glycans in chicken ovalbumin following derivatization of the oligosaccharides by the cyanine dyes. The results were nearly independent of the type of MALDI matrix that was used. Overall, the sensitivity of the MALDI-TOFMS analysis was enhanced ~500-fold with the cyanine dyes. This sensitivity is ~25× better than that obtained with derivatization by Girard’s T reagent.
Improved sensitivity. Structures of cyanine hydrazides used to label oligosaccharides for enhanced detection by MALDI-TOFMS.
The new approach enables detection of subfemtomole levels of oligosaccharide by MALDI-TOFMS. The researchers suggest that cyanine dyes could be useful for protein glycosylation analysis in largescale proteomics studies. (Anal. Chem. 2004, 76, 4537–4542)
Proteome analysis of ovarian epithelial cancer David Lubman and colleagues at the University of Michigan have carried out extensive proteomic analyses of ovarian epithelial cancer. Ovarian epithelial cancer is very hard to detect at its onset and usually is only diagnosed once it is widespread. Lubman and colleagues used a 2-D liquid-phase separation/mass-mapping technique to identify ovarian cancer biomarkers. They also carried out MALDITOFMS/MS peptide sequencing to identify proteins. Two cell lines of ovarian surface epithelium and two adenocarcinoma cell lines were used to compare the protein content of normal and cancerous cells. The investigators found that while many of the proteins showed comparable expression levels in the different cell lines, several proteins were distinctly different. Differences included overexpression of certain proteins, appearance of new proteins, or changes in posttranslational modifications on the proteins. Several of the identified proteins, such as oncoprotein 18/stathmin, were known cancer markers, verifying the proteomic analysis. Lubman and colleagues suggest that databases of proteomic changes in ovarian cancer could be valuable in identifying key biomarkers and understanding the progression of the disease. (Proteomics 2004, 4, 2476–2495)
Agarose stamps for protein microarrays George Whitesides and colleagues at Harvard University have developed a rapid method for patterning microarrays and gradients of proteins using agarose gel stamps. The microfabricated stamps contain an array of posts (50–1000 µm in diam), to which solutions of proteins are added. Either the same or different proteins can be used. For a single protein, the stamps can handle a density of up to 2500 spots/cm2. Once the stamp is “inked” with the protein solutions, the protein spots can be quickly transferred onto a flat substrate such as a glass slide. The pattern
stamping can be repeated >100 for 1 protein and 20 for up to 8 proteins without the need for re-inking. The new approach offers several advantages. The hydrogels are biocompatible, the proteins do not have to be dried prior to stamping, and only subnanomolar levels of proteins are required. In addition, gradients of proteins can be patterned by taking advantage of the tendency for two proteins to diffuse toward each other in the agarose gel. The researchers suggest that the technique could be useful for screening proteinbinding interactions for cases in which the amount of protein is limited. (Proteomics 2004, 4, 2366–2376)
Plant-specific proteins With the complete genome sequence of the plant model organism Arabidopsis thaliana in hand, Rodrigo Gutiérrez and colleagues at Michigan State University and the University of Delaware set out to characterize proteins that are likely to be plant-specific. They identified 3848 A. thaliana proteins, more than half of which have no known function. Membraneassociated proteins and those targeted at mitochondria and chloroplasts were found to be the most poorly characterized. The study shows how little is known about processes that are unique to plants. Even for A. thaliana, the most widely studied plant model, only ~10% of its genes have been characterized experimentally. (Genome Biol. 2004, 5:R53)
Lots of spots. Arrays of fluorescently labeled (a) bovine serum albumin printed onto a hydrophobic surface and (b) collagen printed onto a glass slide. Both patterns were obtained using agarose gel stamps. (Adapted with permission. Copyright 2004 John Wiley & Sons.)
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