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J Biomol Tech. 2008 April; 19(2): 140–144.
PMCID: PMC2361166

Article Watch

This column highlights recently published articles that are of interest to the readership of this publication. We encourage ABRF members to forward information on articles they feel are important and useful to Clive Slaughter, Hartwell Center, St. Jude Children’s Research Hospital, 332 North Lauderdale St., Memphis TN 38105-2794. Tel: (901) 495-4844; Fax: (901) 495-2945: email: gro.edujts@rethgualS.evilC or to any member of the editorial board. Article summaries reflect the reviewer’s opinions and not necessarily those of the Association.

DNA CHARACTERIZATION AND GENOTYPING

Fredlake CP, Hert DG, Kan C-W, Chisel TN, Root BE, Forster RE, Barron AE. Ultrafast DNA sequencing on a microchip by hybrid separation mechanism that gives 600 bases in 6.5 minutes. Proceedings of the National Academy of Sciences, U.S.A. 105;2008:476–481.

While new pyrosequencing-based approaches to DNA sequencing provide very high throughput through parallel processing, read-lengths are typically short (100–230 bases), and achieve accuracy only through high multiplicity of reads from the same sequence. These characteristics are not favorable for sequencing individual templates, or characterizing large repeat-rich regions. For such applications, Sanger sequencing remains the method of choice because of the long reads attainable (>600–700 bases). The present paper describes a chip-based system using electrophoretic conditions modified to produce 600-base reads in as little as 6.5 minutes—a two-thirds reduction in time compared to previous platforms. The traditional linear polyacrylamide sequencing matrix is replaced by a polymer providing an entangled network that is believed to allow electromigration of DNA by alternating reptation through intact polymer, and disruption of network entanglements and dragging of network polymers through the solution in the wake of DNA molecules. Covalently bound channel coatings are replaced by a physically adsorbed coating that affectively minimizes electroosmotic flow and interactions between DNA molecules and the channel walls. Further improvements in the platform are anticipated, and may be expected to yield further substantial improvements in cost and throughput to the Sanger method.

Wong KM, Suchard MA, Huelsenbeck JP. Alignment uncertainty and genomic analysis. Science 319;2008:473–476.

In comparative genomics, large sets of gene sequences are compared in order to deduce phylogenetic history, estimate substitution rates, and identify sites subject to selection pressure. However, the alignments upon which such calculations are based may themselves be subject to appreciable uncertainty, and may strongly influence the outcome of comparative studies. The authors illustrate these problems by presenting the diverse and contradictory conclusions drawn from alignments calculated by seven different programs in a study of amino acid sequences falling into 1502 sets of orthologous gene products from seven yeast strains. While the effects of alignment uncertainty may not be problematic for phylogenetic studies in which genes are carefully selected for consideration based on their ease of alignment, the problem is acute in comparative genomic studies, because many of the most interesting genes, such as those with a high incidence of non-synonymous substitutions, are for that very reason difficult to align. The authors advocate taking alignment uncertainty into account by treating the alignments themselves as random variables and considering the inferences drawn from them in proportion to the varying probabilities that the alignments are correct.

MACROMOLECULAR SYNTHESIS

Lemke EA, Summerer D, Geierstanger BH, Brittain SM, Schulz PG. Control of protein phosphorylation with genetically encoded photocaged amino acid. Nature Chemical Biology 3;2007:769–772.

Investigators here biosynthetically introduce into proteins serine with a photocleavable protecting group. The protecting group is cleaved by irradiation with low-energy blue light, thereby rendering the protein susceptible to in vivo phosphorylation at that site. They introduce 4,5-dimethoxy-2-nirobenzylserine into the yeast transcription factor, Pho4, at a site that controls nuclear export of the protein, influencing the cellular response to inorganic phosphate. The export kinetics of Pho4-GFP constructs are monitored in real time after laser irradiation, and different kinetics are documented for different mutants. The methodology is anticipated to be useful for studies of the role of free serine residues in catalysis, ion transport, and molecular recognition.

MASS SPECTROMETRY

Demeure K, Quinton L, Gabelica V, De Pauw E. Rational selection of the optimum MALDI matrix for top-down proteomics by in-source decay. Analytical Chemistry 79;2007:8678–8685.

In-source decay (ISD) of proteins in MALDI to produce c- and z-ions is an effective method for acquiring amino acid sequence information, which may sometimes be extensive. MALDI matrices differ in their ability to support ISD. 2,5-Dihydroxybenzoic acid is very effective, and α-cyano-4-hydroxycinnamic acid much less so. The authors of the present paper argue that because positive-ion ISD is mediated by transfer of hydrogen radicals from the matrix to the analyte in the MALDI plume, a search for compounds with enhanced hydrogen donor ability might produce a MALDI matrix with superior ISD performance. They identify 1.5-diaminonaphtalene (1,5-DAN) as such a compound, and show it to provide enhanced peptide/protein ISD spectra. Picolinic acid (PA) is also found to be a superior hydrogen donor, and although it is unsuitable as a matrix for peptide/protein ionization on its own, it provides further enhancement of ISD spectra obtained with 1,5-DAN when used as an additive. These matrices will be useful for top-down protein characterization by MALDI.

Wang X, Huang L. Identifying dynamic interactions of protein complexes by quantitative mass spectrometry. Molecular and Cellular Proteomics 7;2008:46–57.

A scheme for stable isotope labeling with amino acids in cell culture (SILAC) labeling is described that distinguishes between proteins interacting stably and proteins interacting transiently. Stable interactions between a bait protein and its binding partners are detected by conventional SILAC. Cells expressing the bait protein with an affinity tag are grown in medium containing isotopically labeled arginine and lysine. Cells expressing the bait protein without a tag (as control) are grown in medium containing arginine and lysine of natural isotopic composition. Labeled or unlabeled amino acids are biosynthetically incorporated into proteins in their respective cultures. Proteins from the two cultures are then mixed together, and purification is performed via the affinity tag. Peptides from proteins that interact stably with the bait protein are recognized by their elevated abundance in isotopically labeled form. However, proteins that interact with the bait protein with high rates of association and dissociation appear like nonspecifically interacting species because their light and heavy forms equilibrate between the tagged and untagged forms of the bait after proteins from the two cultures are mixed together and the purification is still in progress. But if the affinity purification is performed quickly, or is performed before the proteins from the two cultures are mixed, then the opportunity to equilibrate is proscribed or precluded, and such dynamically interacting proteins can be recognized by their perturbed signal strength ratios. This approach is validated in experiments to identify proteins that interact transiently with the proteasome. Interest in this extension of the SILAC method derives from the physiological importance of transient interactions.

Smith DP, Giles K, Bateman RH, Radford SE, Ashcroft AE. Monitoring copopulated conformational states during protein folding events using electrospray ionization–ion mobility spectrometry–mass spectrometry. Journal of the American Society for Mass Spectrometry 18;2007:2180–2190.

Ion mobility mass spectrometry is capable of separating populations of protein molecules according to conformational state and charge state, and is therefore a technique of latent value in the study of protein folding/unfolding, protein aggregation, and the processes leading to protein misfolding diseases. In this paper, ion mobility mass spectrometry is used to study the amyloidogenic protein, β2-microglobulin, that forms fibrils upon unfolding, a process involved in the disease, dialysis-related amyloidosis. Different partially unfolded states that arise upon acid treatment of β2-microglobulin are distinguished, and evidence that these ions are not simply a result of increased protonation of the native state is supplied. An amyloidogenic mutant form of the protein is further shown to produce ion mobility spectra at near-neutral pH, similar to the spectra observed with the wild type at acid pH.

Northen TR, Yanes O, Northen MT, Marrinucci D, Uritboonthai W, Apon J, Golledge SL, Nordström A, Siuzdak G. Clathrate nanostructures for mass spectrometry. Nature 449;2007:1033–1037.

A new desorption/ionization method is introduced in this article. A nanostructured surface with 10-nm pores is prepared by etching of silicon, and these pores are used to trap “initiator” molecules. Sample is then applied on top of the surface thus treated. Local heating of the surface by laser irradiation or a secondary ion beam results in vaporaization of the initiator and desorption of molecules and ions from the overlying sample. Data acquired with the use of perfluorinated siloxanes as initiators are presented, while changing to nonfluorous initiators is shown to alter the ion profiles observed, possibly as a result of differential solubility of different analyte molecules in the initiator. Spectra are dominated by small molecules such as phospholipids and metabolites. The method shows promise for matrix-free imaging.

PROTEINS—PURIFICATION AND CHARACTERIZATION

Rich RL, Cannon MJ, Jenkins J, Pandian P, Sundaram S, Magyar R, Brockman J, Lambert J, Myszka DG. Extracting kinetic constants from surface plasmon resonance array systems. Analytical Chemistry 373;2008:112–120.

Next-generation surface plasmon resonance–based biosensors provide the capability to monitor large numbers of separate binding interactions simultaneously. The ability of one such system, the Flexchip from Biacore, to provide precise and reproducible quantitative information is investigated in this paper. Rate constants are measured for the binding of IgG to immobilized protein A/G under both reaction-limited and mass transport–limited conditions. Because of analyte dispersion, the measured mass transport rate is found to vary with spot position within the flow cell. This necessitates fitting the mass transport rate independently to each spot position when analyzing kinetic data. Spot-to-spot variation in binding kinetics is measured to be less than 9%. The results indicate that simultaneous quantification of hundreds of interactions is rendered possible with such systems.

Structural Genomics Consortium, Architecture et Fonction des Macromolécules Biologiques, Berkley Structural Genomics Center, China Structural Genomics Consortium, Integrated Center for Structure and Function Innovation, Israel Structural Proteomics Center, Joint Center for Structural Genomics, Midwest Center for Structural Genomics, New York Center for Structural GenomiX Research Center for Structural Genomics, Northeast Structural Genomics Consortium, Oxford Protein Production Facility, Protein Sample Production Facility, Max Delbrück Center for Molecular Medicine, RIKEN Structural Genomics/Proteomics Initiative & SPINE2-Complexes. Protein production and purification. Nature Methods 5;2008:135–146.

This paper reviews the collective experience of several groups in purifying more than 10,000 different proteins. The methods that are most frequently successful in the purification of recombinant proteins are listed. Recommendations include initially settling on the use of just one affinity tag, but exploring 10 or more constructs with different N- and C-termini in order to increase the chance of producing a correctly folded protein. Selection of domain boundaries is critical. Alternative tags are not as important in the initial stages. Experience indicates that only 10% of eukaryotic proteins are expressed in soluble form in E. coli under initial screening conditions, so the effort to produce a soluble product is usually substantial. Purification via a His-tag is recommended for most purposes, and purification by gel filtration chromatography, followed by ion exchange chromatography if necessary, is normally effective. This review will provide a helpful resource for those entering the field in deciding which methods to try first.

Alber F, Dokudovskaya S, Veenhoff Lm, Zhang W, Kipper J, Devos D, Suprapto A, Karni-Schmidt O, Williams R, Chait BT, Rout MP, Sali A. Determining the architectures of macromolecular assemblies. Nature 450;2007:683–694.

This article describes a strategy for ascertaining the architecture of a large molecular assembly: the nuclear pore complex. In yeast, this structure has a molecular weight of 50 MDa, and contains at least 456 protein molecules comprising the products of some 30 different genes. The data upon which structural models are based include a component list from affinity purification and protein identification technology; the stoichiometry of each component from quantitative immunoblotting; definition of protein contacts by co-immunoprecipitation; the size and shape of each component from analytical ultracentrifugation; the size, shape, and symmetry of the complex from electron microscopy and cryo-electron microscopy; and the localization of each component within the complex by immuno-electron microscopy. The diverse yet synergistic information from these analyses is translated into spatial restraints, and an ensemble of structures satisfying these restraints is calculated. The ensemble is finally evaluated to produce a structural model. A similar integrative approach is suitable for architectural analysis of other macromolecular assemblies.

PROTEOMICS

Keshishian H, Addona T, Burgess M, Kuhn E, Carr SA. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution. Molecular and Cellular Proteomics 6;2007:2212–2229.

Methods based on multiple reaction monitoring and stable isotope dilution are described here for the quantification of proteins in biological fluids at levels in the low ng/mL range. Such low levels are typical for putative clinical biomarkers, but have generally been beyond the range of mass spectrometric quantification methods. The methodology involves depletion of abundant serum proteins, and minimal fractionation of tryptic peptides by strong cation exchange chromatography prior to reverse-phase chromatography with on-line multiple reaction monitoring-MS. Procedures for establishing accuracy and specificity of the quantitative assays are described in detail.

Usaite R, Wohlschlegel J, Venable JD, Park SK, Nielsen J, Olsson L, Yates JR III. Characterization of global yeast quantitative proteome data generated from wild-type and glucose repression Saccharomyces cerevisiae strains: The comparison of two quantitative methods. Journal of Proteome Research 7;2008:266–275.

Two methods for quantifying the relative levels of proteins during multidimensional protein identification are compared—namely, spectral counting and biosynthetic labeling with stable isotope–labeled amino acids. The comparison is performed in the context of an analysis of four strains of yeast differing only in the presence of components of the Snf1 kinase complex. These strains are closely similar in their protein expression profiles, differing in only 350 proteins out of a total of 2388 quantified. Quantification by spectral counting is substantially improved by eliminating peptides that are shared between two or more proteins. Stable isotope labeling is more sensitive in detecting expression differences, and shows higher precision for proteins of low abundance (i.e., proteins to which few peptides are assigned). Nevertheless, the data indicate that spectral counting is adequate for experiments in which simple protein mixtures are being analyzed or spectra from multiple analyses are available.

MICROARRAYS

Pandya GA, Holmes MH, Sunkara S, Sparks A, Bai Y, Verratti K, Saeed K, Venepally P, Jarrahi B, Fleischmann RD, Peterson SN. A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip whole-genome resequencing platform. Nucleic Acids Research 35;2007:e148.

Resequencing through the use of tiling microarrays has emerged as an important method for genotyping microorganisms. However, deletions in sample DNA relative to the reference sequence can result in poor hybridization and may produce no-calls or false-positive calls in the typing of SNPs. Furthermore, cross-hybridization can produce false-positive SNP calls, and the local destabilizing effect of genuine SNPs can result in false-positive calls at adjacent locations in the genome. Bioinformatic filters are here described to address these errors. For bacterial genomes, the use of these filters enables whole genome amplification to be used instead of PCR amplification to render the procedure more cost-effective and capable of higher throughput. The methodology is applied to a resequencing array for the genome of the gram-negative bacterium Francisella tularensis. Ninety-one percent of false-positive SNP calls are eliminated, with the loss of only 10.7% of true positives. The final base-calling accuracy is 99.992%.

He M, Stoevesandt O, Palmer EA, Khan F, Ericsson O, Taussig MJ. Printing protein arrays from DNA arrays. Nature Methods 5;2008:175–177.

Here is a proof-of-principal experiment demonstrating that a protein array can be manufactured by coupled transcription and translation from a DNA array. A slide to which PCR-amplified fragments are covalently immobilized is interfaced with a second slide derivatized with a surface for capturing in situ synthesized proteins labeled with a tag. Between the two slides is a permeable membrane that carries a cell-free lysate for transcription/translation. Twenty copies of the protein array can be made from a single DNA array. This methodology for the manufacture of protein microarrays circumvents problems associated with the expression, purification, and spotting of large numbers of individual proteins, and eliminates the need for long-term storage of arrays.

FUNCTIONAL GENOMICS AND PROTEOMICS

Silva JM, Marran K, Parker JS, Silva J, Golding M, Schlabach MR, Elledge SJ, Hannon GJ, Chang K. Profiling essential genes in human mammary cells by multiplex RNAi screening. Science 319;2008:617–620.

Schlabach MR, Luo J, Solimini NL, Hu G, Xu Q, Li MZ, Zhao Z, Smogorzewska A, Sowa ME, Ang XL, Westbrook TF, Liang AC, Chang K, Hackett JA, Harper JW, Hannon GJ, Elledge SJ. Cancer proliferation gene discovery through functional genomics. Science 319;2008:620–624.

These papers introduce methodology for screening members of small hairpin RNA (shRNA) libraries for loss-of-function phenotypes on an unprecedented scale. Previous studies of shRNA function have relied upon laborious one-by-one screening or else a strategy of enrichment of pools that restricts the range of phenotypes that can be studied. The present approach relies instead upon depletion from pools, thereby allowing high-throughput screening with respect to a broader range of phenotypes. Pools of up to 20,000 shRNAs are packaged into viral vectors under the control of a promoter, which, upon infection at the level of only one integrated virus per cell, efficiently and stably suppresses expression of the cognate target gene. Each shRNA in the library occupies approximately 1000 cells. It is recognized via a random 60-nucleotide barcode. After culturing the infected cell population for varying periods of time, DNA is prepared, amplified, and allowed to hybridize to a microarray in competition with a common reference to measure depletion of each shRNA in the library. This procedure is used to identify genes important for proliferation and survival in general, and to distinguish cell types (e.g., tumor and normal) on the basis of which genes they are most sensitive to the suppression of. These are the genes marked for target discovery in the design of specific cancer therapies.

Zhang X, Guo C, Chen Y, Shulha HP, Schnetz MP, Laframboise T, Bartels CF, Markowitz S, Weng Z, Scacheri PC, Wang Z. Epitope tagging of endogenous proteins for genome-wide CHIP-chip studies. Nature Methods 5;2008:163–165.

A variety of analytical methods depend on the availability of antibodies against target proteins, including Western blotting, immunoprecipitation, immunofluorescence, and chromatin imunoprecipitation-microarray. The practice of introducing epitope tags against which antibodies are readily available is, of course, widespread. This paper presents a method for such tagging without the need for transgenic expression of recombinant proteins. It relies on introducing DNA encoding the tag sequence by homologous recombination. This “knock-in” is achieved with a vector containing two multiple cloning sites, a sequence encoding a triple-Flag epitope, a neomycin gene flanked with laxP sites, and two inverted terminal repeats. The method is applicable to several human somatic cell lines.

Bantscheff M, Eberhard D, Abraham Y, Bastuck S, Boesche M, Hobson S, Mathieson T, Perrin J, Raida M, Rau C, Reader V, Sweetman G, Bauer A, Bouwmeester T, Hopf C, Kruse U, Neubauer G, Ramsden N, Rick J, Kuster B, Drewes G. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nature Biotechnology 25;2007:1035–1044.

The human genome encodes approximately 500 protein kinases and more than 2000 other purine-binding proteins. Drug lead compounds directed at the ATP-binding sites of kinases are therefore unlikely to be specific for a single kinase. Off-target interactions are presently assessed with panels of recombinant enzymes and model systems. This paper, however, proposes a new approach in which the affinities of a substantial proportion of endogenously expressed kinome members are quantified in parallel. The method employs “kinobeads”—beads to which a panel of seven nonselective kinase inhibitors that bind competitively with ATP are covalently attached. These beads affinity-capture an approximate total of 300 protein kinases from mouse and human cell lines and tissues along with more than 600 additional proteins. The relative affinities of proteins (from K562 cells) for three ABL inhibitors—imatinib (Gleevec), dasatinib (Sprycel), and bosutinib—are measured by supplying varying concentrations of the drugs as competitors with the affinity matrix, and measuring the relative quantities of the proteins bound by means of iTRAQ isobaric tags. The method identifies both known drug targets (ABL and SRK family kinases), and previously unknown, off-target interactions. The method is expected to be useful in drug discovery and in translational studies of drug interactions in patient tissues.


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