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J Biomol Tech. 2005 September; 16(3): 285–293.
PMCID: PMC2291735

Article Watch

AMINO ACID ANALYSIS AND PROTEIN SEQUENCING

Adams CM, Zubarev RA. Distinguishing and quantifying peptides and proteins containing D-amino acids by tandem mass spectrometry. Analytical Chemistry 77;2005:4571–4580. [PubMed]

Serafin SV, Maranan R, Zhang K, Morton TH. Mass spectrometric differentiation of linear peptides composed of L-amino acids from isomers containing one D-amino acid residue. Analytical Chemistry 77;2005:5480–5487. [PubMed]

Two groups present data showing that quantitation of D-amino acids at specified sequence locations in diasteriomeric peptide mixtures is possible by tandem mass spectrometry. Collision-induced dissociation (CID) and electron capture dissociation (ECD) both produce product ion spectra that reveal quantitative differences between the product ions derived from different diasteriomeric peptide forms. These differences are of sufficient magnitude to allow Adams and Zubarev to report accuracies of 1% for ECD and 3–5% for CID in position-specific measurements of D-amino acid content. This work will be of interest both to investigators practicing peptide synthesis and to those researching disease-related phenomena in which racemization plays a part.

DNA CHARACTERIZATION AND GENOTYPING

Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, Wang MD, Zhang K, Mitra RD, Church GM. Accurate multiplex Polony sequencing of an evolved bacterial genome. Science 309;2005:1728–1732.

Margulies M, et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437;2005:376–380. [PubMed]

These two papers represent progress toward the replacement of classical sequencing technology involving bacterial vectors and Sanger sequencing by cheaper, faster, non-electrophoretic methods that have the greater accuracy needed for resequencing studies of variation between individuals and strains. Both groups clonally attach DNA to beads to avoid subcloning in bacteria or handling clones separately, and perform template amplification in an emulsion of beads to decrease reagent consumption. Margulies et al. then use a fiber optic slide of individual wells to read sequences on each bead, whereas Shendure et al. describe an imaging technique involving a conventional epifluorescence microscope that other laboratories can build with off-the-shelf equipment. Shendure et al. resequence a strain of E. coli with an error rate less than one per million consensus bases and a cost less than one-tenth that of conventional sequencing. Margulies et al. sequence the genome of Mycoplasma genitalium with 96% coverage at 99.6% accuracy in one run. Neither method yet achieves read lengths sufficient to sequence mammalian genomes, but improvements are anticipated, and the goal of bringing affordable methods for sequencing entire mammalian genomes to small laboratories appears achievable.

CARBOHYDRATES, GLYCOLIPIDS, AND GLYCOPROTEINS

Qiu R, Regnier FE. Use of multidimensional lectin affinity chromatography in differential glycoproteomics. Analytical Chemistry 77;2005:2802–2809. [PubMed]

Quantitative differences in the extent of addition of specific carbohydrate moieties to specific sites on glycoproteins are made amenable to measurement by the methodology described in this report. Proteins from samples to be compared are first digested with trypsin. The procedure then couples lectin affinity for selection of peptides possessing particular carbohydrate moieties with stable isotope tagging for mass spectrometric quantitation of the glycopeptides. Differential tagging is performed by reaction of peptides with heavy and light forms of N-acetoxysuccinimide. In the present paper, the methodology is applied to comparisons of the relative degree of sialylation of serum glycoproteins using a lectin column from Sambucus nigra agglutinin for carbohydrate selection.

MACROMOLECULAR SYNTHESIS

Fuhrmann M, Oertel W, Berthold P, Hegemann P. Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage. Nucleic Acids Research 33;2005:e58. [PubMed]

When synthesizing an intact gene by assembly of shorter oligonucleotides, the likelihood of encountering an error in the assembled sequence is an additive function of the incidence of incorrect sequences in the component oligonucleotides. An enzymatic method is here described for correcting errors in the assembled gene. Beginning with double-stranded polynucleotide, E. coli endonuclease V or T4 endonuclease VII are used for mismatch deletion. The short overhangs thus generated dissociate at the reaction temperature of 37°C, and the resulting single-stranded DNA is degraded by a 3′–5′-exonuclease such as E. coli exonuclease I. This procedure is successful in removing deletions and insertions from the primary ligation product, and diminishes the incidence of errors in the cloned gene.

Palonić DP, Ide ND, van der Donk WA, Gin DY. Aziridine-2-carboxylic acid-containing peptides: Application to solution- and solid-phase convergent site-selective peptide modification. Journal of the American Chemical Society 127;2005:7359–7369. [PubMed]

A solid-phase synthesis method for incorporating aziridine carboxylic acid into peptides is described. This strongly electrophilic residue allows conjugation with various thiol nucleophiles, including anomeric thiol nucleophiles, farnesyl thiol, and biochemical tags. These may be added either in solution or on the solid support. The procedure provides a general method for site-selective, stereo-selective peptide modification.

Hino N, Okazaki Y, Kobayashi T, Hayashi A, Sakamoto K, Yokoyama S. Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid. Nature Methods 2;2005:201–206. [PubMed]

Suchanek M, Radzikowska A, Thiele C. Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells. Nature Methods 2;2005:261–267. [PubMed]

These articles apply photo-cross-linking to investigation of protein-protein interactions in vivo. Hino et al. use an expanded genetic code to incorporate the photoreactive amino acid p-benzoyl-L-phenylalanine into selected proteins. Suchanek et al. perform biosynthetic incorporation of methionine, leucine, or isoleucine analogs that contain a photoactivatable diazirine ring, which yields a reactive carbene upon light-induced loss of nitrogen. The latter strategy circumvents the need for the production of a bacterial strain with modified components for protein synthesis and overcomes the restriction of incorporating the modified residue into a single site on a single protein.

Carrigan CN, Imeriali B. The engineering of membrane-permeable peptides. Analytical Biochemistry 341;2005:290–298. [PubMed]

Reversible lipid attachment is evaluated as a means to import peptides into cells. Cysteine dodecane disulfide or tyrosine serine myrisate ester is incorporated into peptides during solid-phase peptide synthesis. The lipohilic group mediates internalization by promoting interaction with the cell membrane. The lipid is cleaved from the peptide inside the cell. Effective uptake also depends on a net positive charge achieved by insertion of lysine residues distributed in regions containing acidic groups. This strategy for promoting cellular uptake of peptides avoids the interactions with cellular components and the tendency to aggregation that characterize peptides with protein transduction domains containing polyarginine sequences.

METABONOMICS

Wilson ID, Nicholson JK, Castro-Perez J, Granger JH, Johnson KA, Smith BW, Plumb RS. High resolution “ultra performance” liquid chromatography coupled to oa-TOF mass spectrometry as a tool for differential metabolic pathway profiling in functional genomic studies. Journal of Proteome Research 4;2005:591–598. [PubMed]

This article exemplifies applications of the enhanced chromatographic capabilities of “ultra performance liquid chromatography” (UPLC). Higher plate counts and peak capacities are realized in UPLC by use of new commercially available stationary phases with reduced particle size (1.7 μm) and a chromatographic system operating at the elevated pressures (12,000 psi) needed to maintain flow through the finely divided particle bed. In the present publication, UPLC is used for LC/MS analysis of urinary metabolites. The peak capacity is more than double that obtained by conventional HPLC using a 3.5-μm stationary phase in separations of comparable duration. Gradient elution times can be reduced from 10 min to 2 min, and although such rapid separations result in reduced resolution, the number of ions detected is still better than that obtained with 10-min separations by conventional HPLC. Mass spectral sensitivity is enhanced by three- to fivefold, in part because the improved separation of ions results in less signal suppression.

MASS SPECTROMETRY

Coon JJ, Ueberheide B, Syka JEP, Dryhurst DD, Ausio J, Shabanowitz J, Hunt DF. Protein identification using sequential ion/ion reactions and tandem mass spectrometry. Proceedings of the National Academy of Sciences USA 102;2005:9463–9468.

Methods are presented for acquisition of sequence information from intact proteins using electron transfer dissociation (ETD) in a modified linear ion-trap mass spectrometer. Fluoranthrene anions are used as the electron donor for ETD. Because the multiply charged protein ions produced by electrospray yield ETD product ions that are themselves multiply charged, such product ion spectra are complex and difficult to interpret. To overcome this difficulty, the ETD product ions are reacted with deprotonated benzoic acid to produce charge reduction by proton transfer to the benzoic acid. The resulting peptide c- and z-ions are predominantly of one charge state and can readily be interpreted in terms of sequence. The overall methodology is applicable on a chromatographic timescale. Fifteen to forty amino acids at both the N- and C-termini of several proteins are sequenced, and post-translational modifications at the N-terminus of a histone protein are elucidated. This work is anticipated to greatly stimulate the use of “top-down” protein identification in proteomics.

Hu Q, Noll RJ, Li H, Makarov A, Hardman M, Cooks RG. The Orbitrap: A new mass spectrometer. Journal of Mass Spectrometry 40;2005:430–443. [PubMed]

A description of the principles of operation, construction, and characteristics of the new Orbitrap mass spectrometer from Thermo Electron is presented. Ions are radially trapped about a central spindle electrode, and mass-to-charge ratios are measured from the frequency of harmonic oscillations in image currents induced by the trapped ions. Mass spectra are computed by Fourier transformation. The device is capable of high resolution (up to 150,000), large space-charge capacity, high mass accuracy (2–5 ppm), a mass-to-charge ratio range of 6000, and a dynamic range of 103. Applicability to characterization of proteins, peptides, oligosaccharides, and metal complexes is demonstrated. The availability of this instrument, combining simplicity with advanced analytical capabilities, is likely to have a considerable impact on biological mass spectrometry.

Van Berkel GJ, Ford MJ, Deibel MA. Thin-layer chromatography and mass spectrometry coupled using desorption electrospray ionization. Analytical Chemistry 77;2005:1207–1215. [PubMed]

This paper describes an application of the important new technique of desorption electrospray ionization (DESI), introduced by Cooks and coworkers in 2004. This is an atmospheric pressure technique in which an electrospray-generated plume of charged droplets is directed at an object and desorbs ions from a surface. Leather and nitrile gloves, a tomato skin, a medicine tablet, and a drop of blood on a finger have all proven suitable substrates for DESI. The present paper describes the application of the technique to coupling thin-layer chromatography and mass spectrometry. Ions are desorbed from reverse-phase thin layer chromatography plates, both in positive- and negative-ion modes. It may be anticipated that many more applications of DESI will soon become available.

Norris JL, Porter NA, Caprioli RM. Combination detergent/MALDI matrix: Functional cleavable detergents for mass spectrometry. Analytical Chemistry 77;2005:5036–5040. [PubMed]

Cleavable detergents whose surfactant properties can be ablated when no longer appropriate by acid cleavage of the polar head group from the apolar tail have been available since 2002, and have found an important place in mass spectrometry. The present paper describes the synthesis and use of a new family of acid-cleavable detergents that liberate α-cyano-4-hydroxycinnamic acid or 3,5-dihydroxycinnamic acid upon cleavage. They thus generate conventional MALDI matrices in situ upon cleavage. Profiling cell lysates, increasing signal-to-noise ratios for many ions, and detection of membrane proteins are described as applications of these reagents.

PROTEINS—PURIFICATION AND CHARACTERIZATION

Wang Y-C, Stevens AL, Han J. Million-fold preconcentration of proteins and peptides by nanofluidic filter. Analytical Chemistry 77;2005:4293–4299. [PubMed]

A simple microfluidic device is used to concentrate proteins as much as 106- to 108-fold. The device consists of a pair of parallel microchannnels, one of which contains the protein, connected by a short nanochannel between them. When an electric field is applied along the cross-nanochannel, an ion-depletion zone is generated. An electric field applied along the microchannel (i.e., in a direction orthogonal to the cross-channnel) then causes electroosmosis and the production of a stable, extended zone of ion depletion in the vicinity of the cross-channel. Protein molecules accumulate in front of the ion-depletion region. When concentration is complete, the protein can be eluted, essentially using the device as an injector. The system is tested with a solution of green fluorescent protein to measure concentrations fluorimetrically. Beginning with a 33 fM solution of the protein, a 107-fold increase in concentration is achieved in one hour. Such a device can be interfaced with a mass spectrometer or with a variety of other analytical systems.

Suh M-H, Ye P, Datta AB, Zhang M, Fu J. An agarose-acrylamide composite native gel system suitable for separating ultra-large protein complexes. Analytical Biochemistry 343;2005:166–175. [PubMed]

A native gel electrophoresis system is described for separation of large multi-protein assemblies. It resolves native, unphosphorylated RNA polymerase Pol II from the hyperphosphorylated Pol IIo. Complexes between Pol II and the regulatory protein Fcp1, a phosphatase specific for the C-terminal repeat domain of Pol II, are also separated from both free Pol II and free Fcp1. The discriminative power of the technique is further demonstrated by separating complexes between Pol II and wild-type Fcp1 from complexes between Pol II and the mutant Fcp1(D172N), which differs by just one negative charge.

Navratilova I, Eisenstien E, Myszka DG. Measuring long association phases using Biacore. Analytical Biochemistry 344;2005:295–297. [PubMed]

Molecular interaction analysis is best conducted at concentrations of the interacting species close to the equilibrium dissociation constant (Kd). For high-affinity systems, this entails working at correspondingly low concentrations. But with such low concentrations, the binding reaction is slow, even if the association rate constant is fast, and the system takes a long time to reach equilibrium. In the Biacore system, the analyte delivery syringe is of limited volume, restricting its ability to sustain prolonged deliveries. Therefore delivery of the analyte for runs of long duration requires use of an external pump. The present article reports analysis of the high-affinity interaction between barnase and barstar (Kd ~ 1.6 × 10−14 − 2.5 × 10−12 M). In this system, a high flow rate is needed to minimize the effects of mass transport, a requirement that is satisfied by delivering barnase with an ancilliary peristaltic pump. Acquisition times as long as 12 hours at undiminished flow rate allow association and dissociation rate constants to be measured accurately.

Park C, Marqusee S. Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding. Nature Methods 2;2005:207–212. [PubMed]

“Pulse proteolysis” in the presence of varying concentrations of denaturant is proposed as a simple method for measuring protein stability that does not require sophisticated biophysical instrumentation or large amounts of protein. The concept is to digest the unfolded protein in an equilibrium mixture of folded and unfolded protein, using an excess of protease and a digestion time that is short compared to the folding/unfolding relaxation time. In the present paper, ΔGunfo is measured for E. coli ribonuclease H and its variants using pulse proteolysis to degrade unfolded protein in the mixture, thus allowing folded and unfolded species to be quantitated. The binding of maltose-binding protein to maltose is also studied by the method. In this system, ligand binding affects both the stability and the kinetics of unfolding.

Brittain SM, Ficarro SB, Brock A, Peters EC. Enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry. Nature Biotechnology 23;2005:463–468.

The term fluorous means “highly fluorinated or perfluorinated.” Tagging a compound with short per-fluoroalkyl moieties enables the tagged molecules to be separated from a mixture by solid-phase extraction over fluorous-functionalized silica gel through the selectivity of fluorine-fluorine interactions. In the present paper, various subsets of peptides are highly enriched using fluorous tags, including peptides bearing different side-chain groups or post-translational modifications (phosphorylation). The method is extremely selective, but can also readily be adapted to enrich different subsets of peptides by exploiting the facile synthesis of perfluoroalkyl chains bearing different reactive functional groups. Moreover, the selectivity of the enrichment procedure can readily be tailored by adjusting the nature of the perfluoroalkyl groups bound to the solid-phase extraction resin. For example, species with different fluorine content can be separated, allowing resolution of peptides with different numbers of phosphorylation sites. Elution is simply performed with solutions of methanol, which is thus fully compatible with mass spectrometry. The method avoids many problems with conventional tags such as biotin, including inefficient recovery of bound species and fragmentation during MS/MS analysis. Fluorous affinity tags are likely to find extensive future use in proteomics.

Zhong H, Marcus SL, Li L. Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/MS for protein identification. Journal of the American Society of Mass Spectrometry 16;2005:471–481.

Lin S-S, Wu C-H, Sun M-C, Ho Y-P. Microwave-assisted enzyme-catalyzed reactions in various solvent systems. Journal of the American Society of Mass Spectrometry 16;2005:581–588.

These papers exemplify the increasing use of microwave irradiaton to accelerate reactions in analytical biochemistry. Zhang et al. perform partial acid hydrolysis of proteins using 25% trifluoroacetic acid. Peptides of a size assignable by MS/MS analysis are produced. With microwave irradiation, hydrolysis reaction times are as short as 10 min. Lin et al. use microwaves for rapid (10 min) proteolysis with trypsin for protein identification by mass spectrometry. The brief incubation period permits the use of reaction conditions that would inactivate the enzyme during prolonged incubations.

PROTEOMICS

Villanueva J, Philip J, Chaparro CA, Li Y, Toledo-Crow R, DeNoyer L, Fleisher M, Robbins RJ, Tempst P. Correcting common errors in identifying cancer-specific serum peptide signatures. Journal of Proteome Research 4;2005:1060–1072. [PubMed]

The Sloan-Kettering group describes its extensive experience in developing a rigorous, reproducible, bias-free peptide-profiling procedure for use in the discovery of biomarkers in serum. The method involves solid-phase extraction of proteins with reverse-phase magnetic beads, followed by MALDI-TOF mass spectrometry. This paper documents variables that represent major sources of bias in such studies, including blood collection, clotting procedure, serum storage and handling conditions, protein extraction methods, conditions for preparation of samples for MALDI, spectral acquisition methods, and signal processing.

Koomen JM, Li D, Xiao L-c, Liu TC, Coombes KR, Abbruzzese J, Kobayashi R. Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery. Journal of Proteome Research 4;2005:972–981. [PubMed]

This study provides sequence assignments for a substantial number of the protein and peptide species (more than 250) detected in heparinized plasma by MALDI-TOF mass spectrometry in the course of bio-marker discovery projects. Using tandem mass spectrometry, it is found that the number of proteins from which these species are derived is very small, approximately 20 in total. They are all common serum constituents—e.g., fibringen, complement components, antiproteases, and carrier proteins. This finding implies that the numbers of proteins being sampled in plasma biomarker discovery programs is strictly limited, and highlights the need to understand the biological processes underlying the proteolysis producing the fragments. Exercising control over such proteolysis will be necessary to ensure reproducible proteomic results from plasma.

Che F-Y, Lim J, Pan H, Biswas R, Fricker LD. Quantitative neuropeptidomics of microwave-irradiated mouse brain and pituitary. Molecular and Cellular Proteomics 4;2005:1391–1405. [PubMed]

Microwave irradiation is used to control proteolysis in brain tissue to increase the yields of neuropeptides (two- to threefold in some brain regions) and diminish the degradation of abundant proteins that tend to overwhelm neuropeptide detection. Irradiation of intact mouse heads is performed immediately after decapitation in a conventional microwave oven. The procedure greatly reduces the levels of protein degradation products. Forty-one neuropeptides and fragments of secretory pathway proteins are identified. The peptides are derived from 15 precursor proproteins.

Zolotarjova N, Martosella J, Nicol G, Bailey J, Boyes BE, Barrett WC. Differences among techniques for high-abundant protein depletion. Proteomics 5;2005:3304–3313. [PubMed]

This paper documents the performance of the polyvalent immunodepletion system offered by Agilent Technologies (Wilmington, DE) for removal of six highly abundant proteins from serum. There is concern that proteins of interest may be lost in this procedure by nonspecific sticking or by direct interactions with albumin. However, conditions are identified under which the bound fraction contains minimal low-molecular-weight species co-binding with the six targeted proteins. By contrast, removal of albumin with Cibacron blue dye results in removal of many proteins of lower abundance from serum.

Karp NA, Lilley KS. Maximizing sensitivity for detecting changes in protein expression: Experimental design using minimal CyDyes. Proteomics 5;2005:3105–3115. [PubMed]

Levels of variation of the same sample across multiple gels are measured for a wide range of biological samples using the DIGE system to test the validity of the assumptions underlying the multivariate statistical tests routinely used to detect proteins with expression changes in 2D electrophoresis studies. The assumption that variation in spot intensities is normally distributed is validated. The assumption that the variance is independent of spot intensity after log transformation is also largely valid: however, some proteins are shown to be exceptional, apparently owing to preferential binding of the Cy3 or Cy5 dye. The number of replicates that must be run to achieve the sensitivity needed to detect expression changes typically sought by investigators is assessed, and a cost-benefit analysis is presented. The merits of greater multiplexing by use of a third dye, Cy2, are also considered.

Tao WA, Wollscheid B, O’Brian R, Eng JK, Li X-j, Bodenmiller B, Watts JD, Hood L, Aebersold R. Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. Nature Methods 2;2005:591–598. [PubMed]

A new chemical strategy is described for enrichment of phosphopeptides, based on use of a dendrimer, a soluble, branched polymer that has functional groups on its surface. Phosphorylated peptides react selectively with amine groups on the dendrimer, producing phosphopeptide-polymer conjugates that are larger than unmodified peptides. The conjugates can therefore be separated from unconjugated peptides using a simple spin column with 5-kDa cut-off. The phosphopeptides are then released from the dendrimer under acidic conditions, and separated from the dendrimer by another round of size exclusion in the spin column. This procedure is used with an initial antiphosphotyrosine protein immunoprecipitation step to identify previously unknown sites of tyrosine phosphorylation on a large number of proteins.

Chalkley RJ, Baker PR, Hansen KC, Medzihradszky KF, Allen NP, Rexach M, Burlingame AL. Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer. Molecular and Cellular Proteomics 4;2005:1189–1193. [PubMed]

This paper presents a detailed analysis of the CID spectra acquired on a QSTAR mass spectrometer in a multidimensional chromatography experiment. Through manual verification of database search results and de novo spectral interpretation, 2368 CID spectra of the 3269 spectra acquired could be assigned with high confidence. The reasons for non-assignment of the remaining 901spectra are documented in detail. They include spectra with insufficient ions to assign manually, spectra not of a peptide, spectra derived from precursors with wrongly assigned precursor charge state or mis-assigned precursor monoisotopic peak, and spectra from nontryptic peptides, among other problems. This study illustrates the need for careful scrutiny of the data to extract maximum benefit from such information-rich data sets.

MICROARRAYS

Zhang J, Finney RP, Clifford RJ, Derr LK, Buetow KH. Detecting false expression signals in high-density oligonucleotide arrays by an in silico approach. Genomics 85;2005:297–308. [PubMed]

Probes on Affymetrix GeneChip arrays are scrutinized for problems that would yield consistent but inaccurate signals across multiple experiments. Probe sequences are mapped to human mRNA sequences, and problematic mappings are evaluated on the basis of quality of matching sequences, consistency of sequence representation in reference databases such as LocusLink and UniGene, and mRNA-genomic sequence alignment. Probe-gene mapping errors due to problematic probe sequences are thus distinguished from incompleteness of reference gene annotation. On the U29A/Av2 array, 20,696 (10.5%) probes are identified as nonspecific (capable of hybridizing to multiple genes) and 18,363 (9.3%) probes as incapable of hybridizing to the target transcript. On the U133A array, the numbers are 29,405 (12.1%) and 19,717 (8.0%), respectively. These probes showed a 20–30% decrease in detecting present signals compared with normal probes in GeneChip experiments, and 30% lower consistency with qualitative SAGE and EST expression data. An application for improving the accuracy of expression analysis based on these results is available at http://masker.nci.nih.gov.

Larkin JE, Frank BC, Gavras H, Sultanna R, Quackenbush J. Independence and reproducibility across microarray platforms. Nature Methods 2;2005:337–343. [PubMed]

To address concerns about the reproducibility of microarray gene expression data across platforms, data from a short oligonucleotide array, the Affymetrix Mouse Genome 430 2.0 GeneChip, are compared to data from a spotted cDNA array in an experiment on angiotensin II-induced hypertension. A total of 11,710 genes are represented on both arrays. The finding is that biological treatment has a far greater impact on measured expression levels than platform for more than 90% of these genes. This result is validated by qRT-PCR. For those genes yielding discrepant results between the two platforms, qRT-PCR generally did not confirm either set of data, suggesting that sequence-specific effects rather than platform differences caused the problem. These results are reassuring that valid cross-platform comparisons are achievable.

Irizarry RA, et al. Multiple-laboratory comparison of microarray platforms. Nature Methods 2;2005:345–349. [PubMed]

Bammler T, et al. Standardizing global gene expression analysis between laboratories and across platforms. Nature Methods 2;2005:351–356. [PubMed]

In the first of these two papers, ten laboratories in the Washington, DC–Baltimore area acquire relative (differential) gene expression data on the same set of samples on three different platforms: (1) Affymetrix GeneChips, (2) two-color, spotted cDNA arrays, and (3) two-color, long oligonucleotide arrays. The variation could be attributed mainly to differences between labs rather than between platforms. In the second paper, a consortium of seven laboratories tests standard samples on a variety of platforms, and finds poor reproducibility between platforms and between laboratories until standardized protocols for RNA labeling, hybridization, microarray processing, data acquisition, and data normalization are agreed upon. These results encourage optimism that cross-platform comparisons are possible, but caution that this can be achieved only with adoption of experimental standards and use of appropriate data filters.

FUNCTIONAL GENOMICS AND PROTEOMICS

Chesler EJ, Lu L, Shou S, Qu Y, Gu J, Wang J, Hsu HC, Mountz JD, Baldwin NE, Langston MA, Threadgill DW, Manley KF, Williams RW. Complex trait analysis of gene expression uncovers polygenic and pleiotropic networks that modulate nervous system function. Nature Genetics 37;2005:233–242. [PubMed]

This paper exemplifies expression genetics studies, which seek to elucidate the genetic basis of gene expression phenomena (embracing genetic differences in both cis- and trans-acting regulatory elements) by acquiring microarray data from individuals in a pedigree or experimental cross and then applying to those data procedures for mapping gene loci for quantitative traits. The present article applies this methodology to analysis of nervous system function. Expression data are gathered with Affymetrix GeneChips from the extensively phenotyped BXD panel of recombinant inbred mouse strains. The finding is that a small number of gene loci jointly modulate large sets of transcripts and neural phenotypes in patterns specific to each brain sub-region. Analytical and graphical tools developed for characterizing genes modulating networks of traits are made available at http://www.webqtl.org/.

Hubner N, et al. Integrated transcriptional profiling and linkage analysis for identification of genes underlying disease. Nature Genetics 37;2005:243–253. [PubMed]

Genes regulating gene expression in the BXH/HBX panel of rat recombinant inbred strains are investigated. The parental strains for this panel are the spontaneously hypertensive rat (SHR) and the normotensive Brown Norway rat (BN). Gene expression data were acquired for fat and kidney, both tissues important for the pathogenesis of hypertension. Many of the loci most strongly associated with expression differences are for cis-regulatory elements, are inherited essentially as monogenic traits, and are good candidate genes for known physiological quantitative trait loci. A set of 73 genes associated with hypertension is presented.

Petti AA, Church GM. A network of transcriptionally coordinated functional modules in Saccharomyces cerevisiae. Genome Research 15;2005:1298–1306.

From the perspective of systems biology, the phenotype of a unicellular organism is described in terms of a network of regulatory relationships between its genes, proteins, and other molecules. Such networks are found to have a modular structure in which components involved in a common subcellular process show more regulatory interactions with one another than they do with components mediating other processes. Nevertheless, the behavior of the system as a whole must depend on inter-module coordination. Although most studies of functional modularity seek to define components of modules, the present article presents an algorithm to automate the detection of module co-regulation and the identification of transcription factors that may mediate it. One hundred seventy-five transcription factors are scrutinized, and the modules are defined according to a standard database. The results indicate extensive module co-regulation, detect previously unknown interactions, and lead to identification of transcription factors that may mediate co-regulation. For example, the transcription factor Gcr1p is proposed to exercise transcriptional coordination of glycolysis and lipid metabolism, and glycolysis and phosphoinositide signaling are suggested to regulate each other reciprocally.

BIOINFORMATICS

Löytynoja A, Goldman N. An algorithm for progressive multiple alignment of sequences with insertions. Proceedings of the National Academy of Sciences USA. 102;2005:10,557–10,562.

In alignment of pairs of sequences, traditional dynamic programming algorithms such as the one by Needleman and Wunsch position gaps in the aligned sequences by simple application of appropriate gap penalties and residue matching scores. Alignment of multiple sequences is usually accomplished heuristically by progressive iteration of pairwise alignments. But these methods do not distinguish between insertions and deletions. They must avoid penalizing single insertion/deletion events multiple times, and do this by invoking compensatory scoring schemes, but the result is artifactual superposition of gaps, with overmatching of sequences and underestimation of the number of insertion/deletion events. In the present paper, a new algorithm is described that distinguishes insertions from deletions and penalizes insertions only once. The algorithm infers a greater number of insertion/deletion events and creates alignments with spatially less concentrated gaps. These calculations may more faithfully reflect the evolutionary changes in the sequences.

Mitchell AA, Chakravarti A, Cutler DJ. On the probability that a novel variant is a disease-causing mutation. Genome Research 15;2005:960–966. [PubMed]

What is the likelihood that a novel genetic variant found in a patient and not in a group of controls is the disease-causing mutation in that patient? Using a method derived from population genetics theory, this paper answers that, in the case of a variant detected in a patient by resequencing 10 kb of genomic DNA, the probability of finding a neutral (i.e., irrelevant) variant in a patient and not in 50 normal controls is only about 0.15, making the discovery a very weak piece of evidence for involvement of the mutation in the disease. The method used in this paper makes a number of simplifying assumptions, including either absence of recombination between SNPs or free recombination, a population of constant size in genetic equilibrium, and the absence of selection. Any or all of these assumptions may not hold in any particular case, with the potential for making the outcome even less favorable for the gene hunter.

Marchini J, Donnelly P, Cardon LR. Genome-wide strategies for detecting multiple loci that influence complex diseases. Nature Genetics 37;2005:413–417. [PubMed]

It is now possible to perform high-throughput genotyping of very large numbers of SNPs. Application of this technology to the search for associations between genes that might contribute to the heritability of complex diseases is imminent. How should the results be analyzed, given that the contribution of common variants to the heritability of a disease might individually be small, yet the statistical penalty for testing vast numbers of hypotheses of interactions between loci may be very large? Is it best to scan the genome for alleles that individually meet stringent statistical tests for disease association, or to evaluate all possible pairs of interactions for significance? This paper shows that interaction-based searches are computationally feasible and that they can be more powerful than single-locus approaches despite the statistical penalty for multiple testing. This may not be true when single-locus effects are large relative to interaction effects, especially if single-locus effects are sufficient to identify at least one of the loci. A useful compromise between the two approaches might be to conduct single-locus tests at low stringency and then examine the loci thus picked for significance of all possible two-way combinations at high stringency, correcting for multiple testing.


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