PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jbtJBT IndexAssociation Homepage
 
J Biomol Tech. 2007 December; 18(5): 336–340.
PMCID: PMC2392990

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

Meyer M, Stenzel U, Myles S, Prüfer K, Hofreiter M. Targeted high-throughput sequencing of tagged nucleic acid samples. Nucleic Acids Research. 2007;35:e97. [PubMed]

Despite the large increase in throughput afforded by 454 sequencing (Margulies et al., Nature 437;2005:376–380) compared to Sanger sequencing, the method is inherently limited in the number of samples that can be sequenced in parallel. This limitation is particularly relevant to population genetic and medical genetic studies. To overcome this problem, a barcoding system based on a ligation procedure similar to that used in conventional 454 library preparation is described. It requires a single oligonucleotide to be synthesized for barcoding each sample. The tag is 6 bp long, and therefore permits 72 samples to be distinguished after re-pooling into a single library. If the 454 sequencing plate is divided into two sections, 144 samples can be sequenced in parallel.

Albert TJ, Molla MN, Muzny DM, Nazareth L, Wheeler D, Song X, Richmond TA, Middle CM, Rodesch MJ, Packard CJ, Weinstock GM, Gibbs RA. Direct selection of human genomic loci by microarray hybridization. Nature Methods. 2007;4:903–905. [PubMed]

Okou DT, Steinberg KM, Middle C, Cutler DJ, Albert TJ, Zwick ME. Microarray-based genomic selection for high-throughput resequencing. Nature Methods. 2007;4:907–909. [PubMed]

Porreca GJ, Zhang K, Li JB, Xie B, Austin D, Vassallo SL, LeProust EM, Peck BJ, Emig CJ, Dahl F, Gao Y, Church GM, Shendure J. Multiplex amplification of large sets of human exons. Nature Methods. 2007;4:931–936. [PubMed]

With the recent introduction of instrumentation capable of very high rates of sequence acquisition at very low cost, multiplexed PCR-based methods for enriching specific regions of complex genomes to use as sequencing templates has become the rate-limiting step in the sequencing process. The three papers cited address the need for more streamlined methods for targeted enrichment for use in resequencing studies. Albert et al. and Okou et al. capture DNA segments by hybridization to microarrays. Albert et al. use custom-designed high-density microar-rays to capture exon-sized gene segments (6726 regions of approximately 500-base length from 660 genes) and locus-sized regions (200 kb to 5 Mb surrounding the human BRCA1 locus). Okou et al. use custom arrays to isolate and resequence 304 kb of DNA within a 1.7-Mb region containing three human loci: FMR1, FMR1NB, and AFF2. Porreca et al. release probes from a custom array and capture and amplify 10,000 human exons in a single mutiplex PCR, although they find that capture efficiency is highly non-uniform. All three groups demonstrate a high level of enrichment specificity. The availability of such methods is expected to find application in numerous clinical and population resequencing studies.

The International HapMap Consortium. A second generation human haplotype map of over 3.1 million SNPs. Nature. 2007;449:851–861. [PubMed]

The results of Phase II of the HapMap Project are summarized in this report. A furtheer 2.1 million SNPs are genotyped in the panel of 270 individuals from four geographic regions used for Phase I. The resulting map contains 3.1 million SNPs, achieves a density of approximately one SNP per kilobase, and is estimated to include 25–35% of the common SNP variation in the populations surveyed. It also improves the representation of rare variation. The data reveal a high incidence of genetic identity over extended chromosomal regions due to the sharing of a common ancestor within 10–100 generations. SNPs that lack association with any other SNP within 100 kb, and therefore lack association with any haplotype, are found to constitute 0.5–1.0% of all high-frequency SNPs. They are usually located near recombination hotspots. The map provides a resource for design and analysis of disease association studies, and promotes a shift from candidate gene approaches toward genome-wide analysis.

Frazer KA, Eskin E, Kang HM, Bogue MA, Hinds DA, Beilharz EJ, Gupta RV, Montgomery J, Morenzoni MM, Nilsen GB, Pethiyagoda CL, Stuve LL, Johnson FM, Daly MJ, Wade CM, Cox DR. A sequence-based variation map of 8.27 million SNPs in inbred mouse strains. Nature. 2007;448:1050–1055. [PubMed]

Yang H, Bell TA, Churchill GA, Pardo-Manuel de Villena F. On the subspecific origin of the laboratory mouse. Nature Genetics. 2007;39:1100–1107. [PubMed]

The genetic diversity available among laboratory mice has long constituted a foundation for pursuit of genetic research into disease susceptibility and cancer biology. Classical laboratory strains owe their diversity to the differences between the four subspecies of Mus musculus that contributed to the gene pool of progenitor “fancy” mice bred in Europe and Asia, while inbred strains derived from wild mice provided further gene sets. Frazer et al. resequence 15 strains of mice—11 classical strains and 4 wild-derived strains—and document 8.3 million SNPs, the vast majority of which are novel. The resulting hap-lotype map reveals considerable regional redundancy of SNP data such that as few as 50,000 dispersed SNPs can be used to deduce the genotype of the remaining 8.3 million, as well as a complete picture of the genetic variation in other strains without the need to resequence them. Yang et al. study the distribution of SNPs throughout the genome and delineate large regions that are identical between the classical strains by reason of common ancestry. These represent blind spots for studies of natural variation and complex traits. Other regions are hotspots of diversity. Only 26% of the estimated total variation that exists among all strains examined segregates in the classical strains, suggesting that new lines with greater diversity and better randomization of variants could be constructed for study of complex traits and diseases. The findings therefore validate the goals of the Collaborative Cross project founded for this express purpose.

MASS SPECTROMETRY

Olsen JV, Macek B, Lange O, Makarov A, Horning S, Mann M. Higher-energy C-trap dissociation for peptide modification analysis. Nature Methods. 2007;4:709–712. [PubMed]

The LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific) provides collision-induced product ion spectra of high resolution and mass accuracy. Methods for extending the use of this instrument to provide information about product ions of low mass are described in this paper. This is achieved by fragmenting ions either in the C-trap normally used to store ions on their way from the linear ion-trap to the orbitrap, or in a special collision cell positioned at the far end of the C-trap that is implemented in a version of the instrument called the LTQ-Orbitrap XL. Detection of immonium ions such as that for phosphotyrosine is demonstrated. Additionally, in a SILAC experiment the instrument is used to isolate precursors using a broad mass window so that both the heavy and light SILAC pairs are fragmented together. In the resulting product ion spectra, ions that do not contain the labeled amino acid at the C-terminus (e.g., b-ions) appear as singlets, whereas ions that do contain the C-terminal residue (e.g., y-ions) appear as doublets, allowing the two ion series to be discriminated readily.

Gatlin-Bunai CL, Cazares LH, Cooke WE, Semmes OJ, Malyarenko DI. Optimization of MALDI-TOF MS detection for enhanced sensitivity of affinity-captured proteins spanning a 100 kDa mass range. Journal of Proteome Research. 2007;6:4517–4524. [PubMed]

To help acquire information about high-mass ions of intact proteins by MALDI-TOF mass spectrometry, this article describes methods for sample preparation, mass spectral acquisition, and data processing optimized for the purpose. Sample preparation is performed using weak cation exchange, C3 reverse-phase, and immobilized metal-ion affinity chromatography magnetic beads. In the Bruker Ultraflex III mass spectrometer, adjustments to the ADC offset and preamplifyer bandwidth enhance the detection of broad signals. Post-acquisition signal processing includes baseline subtraction using a decaying exponential model, and integrative down-sampling to achieve a constant number of time steps per fwhm (approximately nine steps in the range up to m/z 100,000). The latter procedure is preferable to moving average integrative filtering, which results in signal broadening. The methodology will assist in the detection of high-mass signals in protein profiling studies.

Ratcliffe LV, Rutten FJM, Barrett DA, Whitmore T, Seymour D, Greenwood C, Aranda-Gonzalvo Y, Robinson S, McCoustra M. Surface analysis under ambient conditions using plasma-assisted desorption/ionization mass spectrometry. Analytical Chemistry. 2007;79:6094–6101. [PubMed]

Several techniques have recently been described for acquiring mass spectrometric information from unprepared surfaces at ambient pressure and humidity. Among these, desorption electrospray ionization (DESI) uses a pneumatically assisted electrospray to desorb ions from the sample surface, and direct analysis in real time (DART) uses a non-thermal plasma from which charged species have been removed and metastable species are directed toward the sample. The present paper describes a new plasma-assisted desorption/ionization (PADI) method, in which a non-thermal radio-frequency-driven atmospheric pressure plasma is directed at the sample surface without removing charged particles. Unlike DART, it uses a class of discharge that operates over a lower power range. Unlike both DART and DESI, the source is cold to the touch and is readily used with thermally sensitive samples. Unlike DESI, PADI requires minimal optimization of operating parameters, does not use solvents, and is less dependent on the angles between the sample and the mass spectrometer inlet and the electrospray/plasma probe and the sample. The new technique is demonstrated by detecting the active ingredients in a variety of nonsteroidal anti-inflammatory and analgesic drugs, and Beecham’s “all in one” cold and flu remedy; and in studies of nicotine in tobacco and thiosufates in garlic. The method is anticipated to find broad application in the pharmaceutical, biological, and forensic sciences.

PROTEINS–PURIFICATION AND CHARACTERIZATION

Song F. A study of noncovalent protein complexes by matrix-assisted laser desorption/ionization. Journal of the American Society for Mass Spectrometry. 2007;18:1286–1290. [PubMed]

Morgner N, Kleinschroth T, Barth H-D, Ludwig B, Brutschy B. A novel approach to analyze membrane proteins by laser mass spectrometry: From protein subunits to the integral complex. Journal of the American Society for Mass Spectrometry. 2007;18:1429–1438. [PubMed]

These two papers provide conditions under which non-covalent protein complexes may be preserved and studied by laser desorption/ionization mass spectrometry. Song uses a conventional UV laser for MALDI mass spectrometry. Soluble protein complexes are incorporated into sinapinic acid matrix by co-crystallization from solution in sinapinic acid and ammonium citrate in water, adjusted to pH 7 with concentrated sodium hydroxide. Organic solvent is omitted from the matrix solution. Morgner et al. study integral membrane proteins. Membrane protein complexes solubilized with detergent (n-dodecyl-β-D-maltoside) are desorbed from buffered aqueous microdroplets using an IR laser tuned to the absorption maximum of water. At very low laser energies, protein complexes are desorbed with small numbers of detergent molecules. At higher laser energies, isolated subunits are observed and detergent molecules stripped off.

Slabinski L, Jaroszewski L, Rodrigues APC, Rychlewski L, Wilson IA, Lesley SA, Godzik A. The challenge of protein structure determination—lessons from structural genomics. Protein Science. 2007;16:2472–2482. [PubMed]

Structural genomics efforts have now provided copious information about both successful and unsuccessful attempts at determination of 3D structure by X-ray crystallography. Building on this information, the present paper reports a “crystallization feasibility” score by which the likelihood that success for any particular target protein will be achieved, and how the protein’s structure might be modified to increase the chance of a successful outcome. Protein features considered in calculating the score include sequence length, isoelectric point, hydropathy, length of disordered segments, and other parameters. Some features correlate with successful protein production, some with successful crystallization, and some with both. Because structural genomics efforts often choose representatives of protein families rather than attempting to study all the family members, the score increases success rates, lowers costs, and shortens the time for structure determination by helping to choose between putative targets.

Goldschmidt L, Cooper DR, Derewenda Zs, Eisenberg D. Toward rational protein crystallization: A Web server for the design of crystallizable protein variants. Protein Science. 2007;16:1569–1576. [PubMed]

Crystallization of proteins is favored by replacing clusters of flexible, solvent-accessible amino acids (e.g., Lys, Glu, Gln) with smaller residues (e.g., Ala) to reduce the conformational entropy of putative contact surfaces. This minimizes the loss of conformational entropy upon crystallization and makes crystal formation more favorable thermodynamically. To aid in the choice of suitable sites to target for mutagenesis in this connection, an algorithm is described that predicts beneficial mutations based on a conformational entropy profile, secondary structure prediction, and sequence conservation. The algorithm is made available on a server called SERp, accessible at http://www.doe-mbi.ucla.edu/Services/SER.

Lawrence MS, Phillips KJ, Liu DR. Supercharging proteins can impart unusual resilience. Journal of the American Chemical Society. 2007;129:10,110–10,112. [PubMed]

This paper explores the possibility of increasing a protein’s solubility and resistance to aggregation by introducing mutations in surface residues, with a view to increasing the net charge on the protein. Eighty percent of naturally occurring proteins have a net charge in the range ±10. Here, green fluorescent protein is mutated to explore net charges in the range –30 to +46. These “supercharged” variants maintain their structural stability and display increased resistance to aggregation upon heating or treatment with a chaotropic agent. Such engineering promises to allow proteins to be modified in ways that suit them for functioning in solvents of choice, resisting aggregation or altering their cellular permeability.

PROTEOMICS

Karp NA, McCormick PS, Russell MR, Lilley KS. Experimental and statistical considerations to avaid false conclusions in proteomics studies using differential in-gel electrophoresis. Molecular and Cellular Proteomics. 2007;6:1354–1364. [PubMed]

Through analysis of same-same data sets acquired with the DIGE system, the authors of this paper demonstrate that Student’s t test is inappropriate for use in three-dye DIGE experiments. In these experiments, two samples labeled with their own distinct dyes are compared against a common internal standard labeled with the third dye. The reason for the problem is that one of the assumptions underlying Student’s t test does not hold for such data—namely, the assumption of random sampling, because standardized abundance values for matched spots calculated with a common standard are inherently non-independent. The variance of the difference between the two groups being compared is a composite of the variance of each sample and the co-variance, but Student’s t test overestimates the true variance by ignoring the covariance, leading to underestimation of the false discovery rate. Two remedies for this problem exist: (1) a significance test that incorporates the co-variance could be used, or (2) the experimental design could be changed to employ a two-dye approach in which each sample is run on a separate gel together with the standard. Similar problems may arise in the context of other multiplexed techniques that employ internal standards, e.g., the iTRAQ system.

Bouyssié D, Gonzalez de Peredo A, Mouton W, Albigot R, Roussel L, Ortega N, Cayrol C, Burlet-Schiltz O, Girard J-P, Monsarrat B. Mascot file parsing and quantification (MFPaQ), a new software to parse, validate, and quantify proteomics data generated by ICAT and SILAC mass spectrometric analysis: Application to the proteomics study of membrane proteins from primary human endothelial cells. Molecular and Cellular Proteomics. 2007;6:1621–1637. [PubMed]

A new software suite named MASCOT File Parsing and Quantification (MFPaQ) is introduced. The software provides an interface to retrieve MASCOT protein lists and sort them according to protein score or other user-defined statistics based on the number and score of peptides identified. It also incorporates an “Extract Daemon,” which extracts intensity lists from raw data. This daemon is written in Visual Basic.Net and is presently compatible with .wiff files generated by the QStar XL and QStarElite mass spectrometers from Applied Biosystems (Foster City, CA). The software calculates peptide ratios and generates non-redundant protein lists for experiments analyzed in multiple searches, and averages and normalizes ratios for listed proteins.

Griffin TJ, Xie H, Bandhakavi S, Popko J, Mohan A, Carlis JV, Higgins L. iTRAQ reagent–based quantitative proteomic analysis on a linear ion-trap mass spectrometer. Journal of Proteome Research. 2007;6:4200–4209. [PubMed]

This paper assesses the effectiveness of the pulsed Q dissociation (PQD) protocol, available for the LTQ linear ion-trap mass spectrometer (Thermo Fisher Scientific), as a means of acquiring quantitative information for iTRAQ-labeled peptides. The protocol enables signals for the low-mass reporter ions from such peptides to be recorded. Optimizing collision energy is found to be essential for acquisition of accurate data. The optimum occurs within a narrow range of values, is instrument dependent, and may change upon routine cleaning or tuning. Increasing the number of microscans and repeat counts enhance accuracy, but reduce the number of peptides identified. Nevertheless, the accuracy of the results is found to approach that obtained with the QStar mass spectrometer, presently the standard instrument for quantification of iTRAQ-labeled peptides. Software for calculating abundance ratios is also described.

Fälth M, Savitski MM, Nielsen ML, Kjeldsen F, Andren PE, Zubarev RA. SwedCAD, a database of annotated high-mass accuracy MS/MS spectra of tryptic peptides. Journal of Proteome Research. 2007;6:4063–4067. [PubMed]

Peptide identification can be performed by directly matching experimental MS/MS data with annotated MS/ MS spectra. This method has proved faster and more accurate than standard methods involving comparison with theoretical spectra. A database of annotated MS/MS spectra for this purpose is announced in this article. The database, called SwedCAD, differs from existing libraries in the high resolution and mass accuracy of the data included. It contains 15,897 different MS/MS spectra of doubly charged, unmodified tryptic peptides. Peptides can be searched according to mass, number of missed cleavages, and sequence motifs. It will serve as a reference for database search algorithms and de novo sequencing algorithms. The data can be downloaded from http://www.bmms.uu.se/CAD/.

FUNCTIONAL GENOMICS AND PROTEOMICS

Korbel JO, Urban AE, Affourtit JP, Godwin B, Grubert F, Simons JF, Kim PM, Palejev D, Carriero NJ, Du L, Taillon BE, Chen Z, Tanzer A, Saunders ACE, Chi J, Yang F, Carter NP, Hurles ME, Weissman SM, Harkins TT, Gerstein MB, Egholm M, Snyder M. Paired-end mapping reveals extensive structural variation in the human genome. Science. 2007;318:420–426. [PubMed]

A method is described for high-throughput identification of structural variants in the human genome (deletions, duplications, insertions, inversions, and complex combinations thereof). The method detects variants measuring 3 kb or larger. It relies on shearing genomic DNA into fragments of approximately 3 kb, circularizing them, randomly shearing them again, and then subjecting the library of re-sheared fragments to 454 sequencing. The distribution of sequence spans relating the paired ends of the re-sheared fragments is then ascertained by computationally mapping the sequences to a reference human genome. Deletions relative to the reference genome are recognized as paired ends spanning a genomic region in the reference genome longer than a specified cut-off. Insertions are recognized as paired ends spanning a region shorter than a specified cut-off. Inversions are detected by a relative orientation different from that in the reference genome. This system is applied to the mapping of structural variants in the genomes of two individuals. Approximately 1300 structural variants are identified in these two individuals, indicating a very high incidence of such variants in human populations. The breakpoint junctions of more than 200 of the variants are determined by pooling PCR products containing affected regions, sequencing them with 454 technology, and then assembling contigs. An average resolution of 644 bp is achieved for breakpoint assignment. Advantages of the methodology are the increase in resolution to a level at which confirmation can be achieved by PCR, and the absence of need to use cloning to prepare a DNA library.

Ju W, Valencia CA, Pang H, Ke Y, Gao W, Dong B, Liu R. Proteome-wide identification of family member-specific natural substrate repertoire of caspases. Proceedings of the National Academy of Sciences, USA. 2007;104:14,294–14,299.

Proteins that behave as substrates for specific prote-olytic enzymes (in this case, caspases) are identified by a procedure called mRNA display. A library of domain-sized proteins is created from poly(A)+ mRNAs under conditions that maintain the linkage between the C-terminus of the translated polypeptide and its cognate mRNA via a puromycin-DNA linker. The N-terminus of the polypep-tide is then biotinylated enzymatically and immobilized on streptavidin-agarose beads. The proteolytic enzyme of interest cleaves the polypeptide, thereby releasing its cognate mRNA, which is then available for amplification for sequencing or iterative rounds of selection. In the present study, 26 known and 89 previously unknown substrates of caspase-3 are identified. The sites of cleavage may be defined by analysis of the proteolytic products. The methodology is applicable to defining the substrate repertoire of a wide variety of proteases.

Bantscheff M, Eberhard D, Abraham Y, Bastuck S, Boesche M, Hobson S, Mathieson T, Perrin J, Raida M, Rao C, Reader V, Sweetman G, Bauer A, Bouwmeester T, Hopf C, Kruse U, Neubauer G, Ramsden N, Rick J, Kuster B, Drewes G. Quantitive chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nature Biotechnology. 2007;25:1045–1044.

This paper suggests a systematic approach to identifying interactions between drugs and off-target cellular constituents. The methodology is applied to the targets of imatinib (Gleevec), an ABL-family kinase inhibitor; and dasatinib (Sprycel) and bosutinib, inhibitors of ABL- and SRC-family kinases. Protein kinases and purine-binding proteins are first captured by binding to immobilized, non-selective kinase inhibitors (kinobeads). ATP-Sepharose has been used for this purpose in the past, but the present investigators employ a resin on which seven different kinase inhibitors are immobilized to capture a broader range of kinases. The drugs of interest are then supplied at varying concentration in solution as competitors for binding of proteins to the resin. Bound proteins are identified and quantified by labeling with iTRAQ isobaric tags. The system also permits drug-induced changes in the phospho-rylation state of the captured proteins to be investigated, and hence components of signaling pathways downstream of the target kinases to be identified. The results confirm known targets of the drugs under test, but also identify novel targets of imatinib.

IMAGING

Choi W, Fang-Yen C, Badizadegan K, Oh S, Lue N, Dasari RR, Feld MS. Tomographic phase microscopy. Nature Methods. 2007;4:717–719. [PubMed]

This paper demonstrates quantitative 3D imaging of refractive index in living cells and tissues (either suspended or substrate attached) without the need to immerse the sample in glycerol or physically rotate the sample in the microscope. The microscope is based on a phase-shifting laser interferometer that provides quantitative phase images from time-dependent interference patterns induced by frequency shifting of the reference beam relative to the sample beam. The angle of sample illumination can be varied with a tilting mirror. 3D images of cultured cells and of C. elegans are presented, and time-dependent changes in cell structure are described. It is anticipated that the technique will find important application in characterizing and correcting for sample-induced aberrations in high-resolution microscopy.


Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities