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J Biomol Tech. 2008 July; 19(3): 225–231.
PMCID: PMC2563919

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.

AMINO ACID ANALYSIS

Sørensen MB, Aaslo P, Egsgaard H, Lund T. Determination of D/L-amino acids by zero needle voltage electrospray ionization. Rapid Communications in Mass Spectrometry 22;2008:455–461.

During studies of the ratio of D- to L-aspartic acid in dentin to measure the age of teeth, these authors discovered that the s/n ratio for the [M+H]+ ion of aspartic acid in electrospray ionization increased 40–50X when the electrospray emitter potential was decreased from the normal 4 kV to 0 V. Increases in detection sensitivity were also noted with other polar amino acids, including Glu, Asn, His, Ser, Asp, Arg, Tyr and Lys. The signal strength measured at 0 V was found to vary with sheath gas flow, eluent flow, and heated capillary temperature. The “zero voltage ESI” method differs from thermospray ionization in that the spray is not preheated, although the ions are desolvated in a heated capillary before reaching the skimmer in the mass spectrometer source. Other classes of small polar molecules may exhibit similar characteristics.

MACROMOLECULAR SYNTHESIS

Schaiberger AM, Moss JA. Optimized sample preparation for MALDI mass spectrometry analysis of protected synthetic peptides. Journal of the American Society for Mass Spectrometry 19; 2008:614–619.

Peptides with protected functional groups are increasingly important as intermediates in “sectional synthesis” of larger peptides. Protected peptides, however, have properties that present difficulties for quality control by MALDI-TOF mass spectrometry. They are sparingly soluble in many solvents, and the presence of protecting groups hinders the detection of the fully protected species so that the relatively strong signals produced by source fragmentation are difficult to distinguish from impurities. Here, a method for sample preparation for protected peptides is described. Peptides are dissolved in dimethyl-formamide. Dithranol is used as MALDI matrix. Matrix is dissolved in a 1:1 acetonitrile/water mixture, and cesium chloride salt is added to form Cs+ adducts with peptides. Peptide is then diluted into the matrix/CsCl solution and dried on a target plate. The method reduces in-source fragmentation and gives improved crystal morphology to yield interpreted data more readily.

Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA III, Smith HO. complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319;2008:1215–1220.

Complete chemical synthesis of a 582,970-bp genome of Mycoplasma genitalium has been accomplished. This genome is the smallest of any known culturable, free-living bacterium. The synthetic genome contains all the genes of wild-type M. genitalium except for the disruption of one gene by an antibiotic marker to block pathogenicity and permit selection. Additionally, sequence “watermarks” were inserted into transposon-tolerant sites to distinguish the synthetic product from naturally occurring genomes. The genome was built by assembling 101 overlapping 5–7-kb DNA cassettes from chemically synthesized oligonucleotides. The cassettes were joined together in genome subassemblies by in vitro recombination, and these subassemblies were cloned as bacterial artificial chromosomes in E. coli. Final assembly of these pieces was performed by transformation-associated recombination cloning S. cerevisiae to yield the completed product as an episome separate from the host genome. It is anticipated that the techniques developed in this project will be useful in producing large DNA molecules containing novel combinations of natural and synthetic DNA segments.

DNA CHARACTERIZATION AND GENOTYPING

Beer NR, Wheeler EK, Lee-Houghton L, Watkins N, Nasarabadi S, Hebert N, Leung P, Arnold DW, Bailey CG, Colson BW. On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets. Analytical Chemistry 80;2008:1854–1858.

Kumaresan P, Yang CJ, Cronier SA, Blazej RG, Mathies RA. High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets. Analytical Chemistry 80;2008:3522–3529.

These two articles illustrate the application of recent advances in digital microfluidics for high sensitivity, high throughput procedures in molecular biology. Both use microfluidic systems in which emulsions of monodisperse, aqueous-in-oil droplets of uniform volume are created to function as tiny reaction vessels that can be individually monitored by fluorescence. Such capabilities enable detection of low copy number species (e.g., pathogens, mutant oncogenes) that amplification bias in traditional PCR methods tends to swamp. Beer et al. demonstrate on-chip isolation and reverse transcription of single RNA molecules with real time fluorescence detection, suggesting the feasibility of quantitative real-time PCR applications. Kumaresan et al. encapsulate individual cells or DNA molecules in droplets together with primer-functionalized beads to prepare DNA for long-read sequencing or genetic analysis. Such capabilities foreshadow large-scale studies of genetic variation in individual cells.

Irizarry RA, Ladd-Acosta C, Carvalho B, Wu H, Brandenburg SA, Jeddeloh JA, Wen B, Feinberg AP. Comprehensive high-throughput arrays for relative methylation (CHARM). Genome Research 18;2008:780–790.

This article contains, firstly, a comparison of the specificity of three commonly used, array-based methods for DNA methylation analysis. In the first, methylated DNA is prepared for hybridization to an array by immunoprecipitation with an antibody. In the second method, methylated DNA is differentially amplified by cutting with methylcytosine-sensitive enzymes and then performing ligation-mediated PCR amplification of the targets. In the third method, methylated DNA is cut with the enzyme McrBC, but no PCR amplification is performed. All three methods were tested in a comparison of the same pair of cell lines: one a highly methylated colorectal carcinoma line, and the other a DNA methyltransferase I and 3B double-knockout line with low levels of methylation. Each method showed limitations. The immunoprecipitation method was found to exhibit poor specificity and limited sensitivity outside CpG islands. The differential amplification method showed incomplete coverage due to its reliance on the sequence specificity of the restriction enzyme employed. The McrBC method was best, but the sensitivity was only 60% at 90% specificity. To overcome this last problem, the authors developed a procedure for the analysis of raw microarray data that makes use of the close correlation of neighboring CpG sites by employing a genome-weighted smoothing algorithm. This procedure improves sensitivity to ~100% at the 90% specificity level.

Harris TD, Buzby PR, Babcock H, Beer E, Bowers J, Braslavsky I, Causey M, Colonell J, DiMeo J, Efcavitch JW, Giladi E, Gill J, Healy J, Jarosz M, Lapen D, Moulton K, Quake SR, Steinmann K, Thayer E, Tyurina A, Ward R, Weiss H, Xie Z. Single-molecule DNA sequencing of a viral genome. Science 320;2008:106–109.

In the continuing rapid advance of single molecule sequencing-by-synthesis technology, methods are reported here for simultaneously sequencing more than 280,000 individual DNA molecules without primer amplification. The method works by polymerase-catalyzed addition of labeled nucleotides to primer-template duplexes immobilized on a surface. Fluorescence imaging is used to monitor nucleotide addition. Because single molecules are being monitored, growth can be asynchronous and forcing each enzymic incorporation step to completion is unnecessary. Read lengths of >30 bases are demonstrated. The method is demonstrated by sequencing the M13 virus to a depth of >150 with 100% coverage. The ability to detect mutations is assessed by incorporating 50 sequence changes (including insertions, deletions, and substitutions in various different contexts) into each of 10 mutated M13 reference genomes. More than 98% of these changes are detected with zero false-positives.

CARBOHYDRATE AND GLYCOPROTEIN ANALYSIS

Vakhrushev SY, Langridge J, Campuzano I, Hughes C, Peter-Katalinic. Ion mobility mass spectrometry analysis of human glycourinome. Analytical Chemistry 80;2008:2506–2513.

The ability of ion mobility separation to simplify spectra of complex analyte mixtures is illustrated in this study of oligosaccharides and glycan conjugates in human urine. Singly, doubly, and triply charged ions are separated in time, facilitating their isolation as precursors for collision-induced dissociation and structural analysis/identification.

METABOLOMICS AND SMALL MOLECULE ANALYSIS

Romanova EV, Rubakhin SS, Sweedler JV. One-step sampling, extraction, and storage protocol for peptidomics using dihydroxybenzoic acid. Analytical Chemistry 80;2008:3379–3386.

Because neural signaling molecules may have very short lifetimes once secreted, there is a need for extraction methods that rapidly terminate the operation of enzymes that degrade them. Here, tissue samples dissected rapidly following sacrifice of an animal are immersed in ≥10X volume of an aqueous solution of 2,5-dihydroxybenzoic acid (DHB) at 20 mg/mL. Peptide mixtures extracted in this way are stable for years without freezing. For MALDI sample preparation, fresh DHB matrix in 50% acetone/50% water is added in large excess (50 mg/mL) to obtain high-quality spectra. An advantage of DHB over 3,5-hydroxycinnamic acid in this procedure is its high solubility; the aqueous solvent promotes extraction of peptides while minimizing lipid contamination and cell membrane disruption. The method is simple and reproducible.

MASS SPECTROMETRY

Luque-Garcia JL, Zhou G, Spellman DS, Sun T-T, Neubert TA. Analysis of electroblotted proteins by mass spectrometry: protein identification after western blotting. Molecular and Cellular Proteomics 7;2008:308–314.

Methodology for handling proteins that have been electroblotted to nitrocellulose membranes is described in this paper. Acetone is used both to dissolve the membranes and simultaneously to precipitate proteins and peptides. For mass analysis of intact proteins, interference due to nitrocellulose is reduced in this way. For protein identification based on mass spectrometry of tryptic peptides, nitrocellulose-bound proteins can be digested on the membrane and peptides thus produced can be precipitated by acetone treatment. Finally, proteins can be identified after Western blotting by removal of the antibodies by washing, as long as a non-protein blocking agent has been used. These procedures may be useful in cases where the involvement of nitrocellulose blotting is unavoidable.

PROTEINS—PURIFICATION AND CHARACTERIZATION

Brady LJ, Valliere-Douglass J, Martinez T, Balland A. Molecular mass analysis of antibodies by on-line SEC-MS. Journal of the American Society for Mass Spectrometry 19;2008:502–509.

Measurement of intact mass of protein pharmaceuticals, e.g., antibodies, provides a wealth of information for quality control purposes. Here, antibodies are prepared for mass analysis by size-exclusion chromatography on-line with an electrospray-TOF mass spectrometer. Chromatography is performed on a polyhydroxyethyl aspartamide column, which can be operated in a size-exclusion mode by using 0.1% formic acid as the mobile phase under isocratic conditions and introducing organic solvent post-column to promote electrospray ionization. The method is rapid (10 min per sample), and is insensitive to sample buffer components, which are removed during size exclusion.

Bagal D, Zhang H, Schnier PD. Gas-phase proton-transfer chemistry coupled with TOF mass spectrometry and ion mobility-MS for the facile analysis of poly(ethylene glycols) and PEGylated polypeptide conjugates. Analytical Chemistry 80;2008:2408–2418.

Polyethylene glycols are commonly conjugated to protein/peptide pharmaceuticals to promote solubility, increase circulating lifetimes, and reduce immunogenicity. For quality control purposes, end group structure, mass of the repeating unit, average molecular weight and molecular weight distribution of the polymer must be demonstrated. Methodology is described in this article to obtain this information by ion mobility mass spectrometry. Because multiple charging of PEGylated proteins in electrospray ionization produces exceedingly complicated mass spectra, gas phase bases are used in the source for charge-stripping to simplify the spectra. In the Waters SYNAPT mass spectrometer, bases can be introduced through the second electrospray inlet supplied with the instrument. Ion mobility separation then permits species with PEGs of different chain length to be resolved. This increases the dynamic range and specificity of the analysis. This application of ion mobility mass spectrometry to an analytically challenging problem highlights the flexibility of the technique.

Froelich JM, Reid GE. The origin and control of ex vivo oxidative peptide modifications prior to mass spectrometry analysis. Proteomics 8;2008:1334–1345.

The oxidation of methionine, S-alkyl cysteine and tryptophan residues during manipulations in vitro can result in significant loss of sensitivity for the detection/identification of peptides containing these residues. The present work documents a greater extent of such oxidation during electrophoresis and in gel digestion than during digestion of proteins in solution. Conditions for reduction of in gel digests prior to mass spectrometry are described: Treatment with 1 M dimethylsulfide/10 M hydrochloric acid for 45 min at room temperature is optimal. Alternatively, if advantage is gained by detecting the oxidized species, for example to confirm the presence of the relevant amino acid by observing side chain cleavage of the oxidized species during MS/MS, then deliberate oxidation is prescribed. Treatment with 30% hydrogen peroxide/5%acetic acid for 30 min at room temperature selectively oxidizes methionine and S-alkyl cysteine.

Jung SY, Li Y, Wang Y, Chen Y, Zhao Y, Qin J. complications in the assignment of 14 and 28 Da mass shift detected by mass spectrometry as in vivo methylation from endogenous proteins. Analytical Chemistry 80;2008:1721–1729.

During a mass spectrometric investigation of the methylation of four proteins, various methylated residues, including Lys, Arg, His, and Glu, were identified by the addition of 14 or 28 Da to peptide mass values. The most commonly used methyl donor for protein methylation is S-adenosyl-L-methionine, and in vivo methylation should be marked by a mass shift if cells are cultured with isotopically labeled methionine as a precursor for this molecule. Surprisingly, however, most of the methylated residues showed no such mass shift. This result indicates either that another methyl donor is being used or that methylation is occurring during sample handling. A high incidence of methylation is ascribed to SDS-PAGE. Isotope labeling is suggested as an important method for validating in vivo methylation in such studies.

Tao L, Kiefer SE, Xie D, Bryson JW, Hefta SA, Doyle ML. Time-resolved limited proteolysis of mitogen-activated protein kinase-activated protein kinase-2 determined by LC/MS only. Journal of the American Society for Mass Spectrometry 19;2008:841–854.

Identifying the fragments produced by partial proteolysis can be highly informative in establishing the domain structure of proteins. The localization of proteolytic cleavage sites simply by measurement of the mass of the proteolytic products may sometimes be rendered ambiguous by limitations in the accuracy of the mass measurements, and additional information such as N-terminal amino acid sequence analysis by automated Edman degradation is frequently needed to resolve ambiguities. The present article, however, demonstrates that mass spectrometric analysis of a time course of digestion may provide sufficient new information to achieve the same end. Larger fragments generated by fewer cleavages early in digestion are easier to assign, and additivity of the mass values of subfragments formed thereafter further constrain possible assignments. The procedure is rapid compared with protocols involving Edman degradation.

Shirai A, Matsuyama A, Tashiroda Y, Hashimoto A, Kawamura Y, Arai R, Komatsu Y, Horinouchi S, Yoshisa M. Global analysis of gel mobility of proteins and its use in target identification. Journal of Biological Chemistry 283;2008:10745–10752.

Here is a large-scale survey of the mobility of Saccharomyces pombe proteins on SDS-PAGE. Of the 4622 proteins tested (representing the products of 93.4% of the predicted structural genes in S. pombe), more than 40% failed to migrate within 10% of the position predicted on the basis of calculated molecular weight (allowing for the additional molecular weight contributed by the poly-His tag used for protein detection by Western blotting). Variation in protein hydrophobicity is the most important contributing factor: Strongly hydrophilic proteins show slower than expected migration. Mobility increases progressively as GRAVY score (hydrophobicity) increases. Isoelectic point also has some influence, with strongly acidic proteins exhibiting smaller than predicted mobility. The behavior of proteins not matching these patterns may be ascribed to post-translational modification. This study shows SDS-PAGE mobility to be a useful method for characterizing the physical/chemical properties of proteins besides their molecular weight.

PROTEOMICS

Brosch M, Swamy S, Hubbard T, Choudhary J. Comparison of Mascot and X!Tandem performance for low and high accuracy mass spectrometry and the development of an adjusted Mascot threshold. Molecular and Cellular Proteomics 7;2008:962–970.

How do commonly used search engines perform when search parameters commensurate with the very high levels of mass accuracy available with contemporary mass spectrometers are selected? Peptide assignment performance of Mascot and X!Tandem is investigated as a function of mass tolerance values typical for low- and high-accuracy data. Both sensitivity and specificity vary widely with changes in mass tolerance settings. However, the effect is more pronounced with Mascot. An approach to the use of high-accuracy data is advocated in which searching is initially performed with broad mass tolerance, and the assignments are then filtered through a narrow mass accuracy window to discriminate correct from incorrect assignments. Sensitivity suffers in this approach, and to regain sensitivity an empirically adjusted Mascot threshold is suggested to allow the user to select the best balance between sensitivity and specificity with both low- or high-accuracy data.

Karp NA, Feret R, Rubtsov DV, Lilley KS. Comparison of DIGE and post-stained gel electrophoresis with both traditional and SameSpots analysis for quantitative proteomics. Proteomics 8;2008:948–960.

The performance of two alternative methods for quantification of proteins by 2-D electrophoresis is compared. In DIGE, proteins are fluorescently labeled before electrophoresis, and pairs of differentially labeled samples are mixed together with a standard sample for co-separation. In the traditional approach, different samples are run on different gels, and the patterns by post-electrophoresis staining are compared. New software marketed by Nonlinear Dynamics, Inc., under the name Progenesis SameSpots, has recently become available for analysis of traditionally stained gels. Spots in the images of all gels to be compared are aligned to the spots on an image selected as the master, and a single spot map and its associated assemblage of spot outlines is employed for the entire gel series for comparative quantification. The problem of missing spot intensity values for spots that go undetected on one or more of the gels in the series is addressed by SameSpots through its use of a common set of spot outlines whether or not a given spot is detected on a particular gel. The result is an increase in the proportion of data pertaining to low spot volumes in conventional gels. No such increase is observed with DIGE because the DeCyder software used for analysis of DIGE patterns stabilizes spot detection areas anyway by comparing patterns from the different dyes on the same gel. A power analysis performed to allow estimation of the relative cost of conducting proteomic analysis by DIGE versus traditional gels with SameSpots analysis indicates that the latter could be more cost-effective for studies analyzing only large changes in abundance, whereas DIGE is more cost-effective when finer discrimination is attempted.

Colzani M, Schütz F, Potts A, Waridel P, Quadroni M. Relative protein quantification by isobaric Silac with immonium ion splitting (ISIS). Molecular and Cellular Proteomics 7;2008:927–937.

Classically, stable isotope labeling with amino acids in cell culture (SILAC) uses metabolic incorporation of labeled lysine and arginine to confer predictable differences in mass between tryptic peptides derived from the proteins in cell cultures to be compared. In the present alternative to this technique, cultures are grown in media containing stable isotope-labeled, yet isobaric, amino acids that can be distinguished by their production of different fragment ions upon collision-induced dissociation. The technique is therefore akin to iTRAQ, except that metabolic incorporation instead of chemical labeling is being utilized. Valine, leucine and isoleucine are each provided in two forms: one labeled with 13C on the backbone carbonyl carbon and the other with 15N on the backbone amide nitrogen. Immonium ions derived from these amino acids are separated by 1 amu because immonium ions lack the carbon atom derived from the carbonyl group, but contain the nitrogen atom derived from the amide group. Ninety-seven percent of tryptic peptides contain at least one residue of Val, Leu or Ile, and peptides that contain more than one can be quantified on the basis of more than one fragment. This approach avoids the proliferation of precursor ion signals encountered with classical SILAC and therefore gains sensitivity. The yield of the requisite immonium ions is promoted in fast scans at elevated collision energy. Sixty-three percent of identified proteins are quantified in the present study. The complete set of culture reagents is cheaper than that for classical SILAC, and the technique can be used for quantification of non-tryptic peptides. A limitation of the new scheme, however, is that it’s not amenable to multiplexing.

Mirzaei H, McBee JK, Watts J, Aebersold R. Comparative evaluation of current peptide production platforms used in absolute quantification in proteomics. Molecular and Cellular Proteomics 7;2008:813–823.

Absolute quantification of peptides is conducted by the method of isotope dilution using stable isotope-labeled peptide standards. Classically, such standards are made individually by chemical synthesis, but, more recently, genes containing concatenated sequences encoding desired sets of standard peptides have been expressed and then digested. The present study compares these two methods with regard to accuracy and handling methods. Twenty-five peptides were chosen and 24 were successfully synthesized chemically. Five genes were synthesized with the coding sequences for the 25 peptides concatenated in different order, and were expressed by in vitro translation using heavy isotope-labeled lysine and arginine. Three were successfully expressed. Tests of various digestion conditions revealed large affects on the results, underscoring the need for optimization. Peptides from in vitro translation were mixed with chemically synthesized peptides and quantified by multiple reaction monitoring. Urea as denaturant for digestion gave the best results, producing peptides mostly in equimolar ratio. The order of the peptides didn’t much affect the yield during digestion, but did affect the success of in vitro translation. Quantification of some peptides was affected by problems with solubilization or stability of the chemically synthesized products. Overall, neither approach was judged to be superior for absolute quantification purposes.

Thingholm TE, Jensen ON, Robinson PJ, Larsen MR. SIMAC (Sequential Elution from IMAC), a phosphoproteomics strategy for rapid separation of monophosphorylated from multiply phosphorylated peptides. Molecular and Cellular Proteomics 7;2008:661–671.

A strategy is developed for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from complex proteome samples. The method is based on sequential elution from IMAC first using acidic conditions (1% TFA, pH 1.0, 20% acetonitrile) to elute monophosphorylated peptide and then basic conditions (ammonia water, pH 11.3) to elute multiply phosphorylated peptides. TiO2 is used after IMAC to remove non-phosphorylated peptides from the pool of monophosphorylated peptides, and to recover mono-phosphorylated peptides from the IMAC flow-through. One advantage of separating mono- and multiply phosphorylated peptides when collision-induced dissociation is employed for peptide identification is that fragmentation schemes differently tailored to these ion types can be utilized. Specifically, monophosphorylated peptides can be subjected to phosphorylation-directed MS3 using MS2 product ions that have undergone neutral loss of one phosphate from the precursor ion, whereas multiply phosphorylated peptides can be subjected to phosphorylation-directed MS3 using MS2 product ions that have undergone loss of two phosphates from the precursor. Using this overall scheme, 306 monophosphorylated peptides, 186 multiply phosphorylated peptides, and 716 different phosphorylation sites are identified from just 120 μg of a tryptic digest of human mesenchymal stem cells, whereas only 232 monophosphorylated peptides, 54 multiply phosphorylated peptides and 350 different sites are identified with an optimized TiO2 method.

MICROARRAYS

Evans-Nguyen KM, Tao S-C, Zhu H, Cotter RJ. Protein arrays on patterned porous gold substrates interrogated with mass spectrometry: detection of peptides in plasma. Analytical Chemistry 80;2008:1448–1458.

The goal of this work is to combine two techniques, protein microarraying and MALDI mass spectrometry, to detect and identify components of biological fluids that specific proteins can bind. To increase the amount of protein in each feature of the microarray, and hence maximize the signal from ligands, a layer of porous gold is deposited on a gold MALDI target by electrolysis to increase effective surface area. Hydrophilic spots are then patterned onto the surface by applying carboxy-terminated self-assembled monolayers with protein array-printing equipment. The surface in between these spots is rendered super-hydrophobic by applying a methyl-terminated self-assembled monolayer that resists fouling by biological fluids by its unwettable character. The purpose of this is to increase amounts of fluid that can be applied to each feature, and to avoid cross-contamination between neighboring features. Antibodies against HA, cMyc, and V5 are used as test proteins and are demonstrated by mass spectrometry to bind their cognate antigens on the microarray. This work is intended to create a platform with sufficient sensitivity to permit screening for new and previously unanticipated ligands for proteins.

FUNCTIONAL GENOMICS AND PROTEOMICS

Poser I et al. BAC TransgeneOmics: A high-throughput method for exploration of protein function in mammals. Nature Methods 5;2008:409–415.

The success of large-scale protein functional analysis in yeast has relied on the use of efficient intrinsic homologous recombination to introduce genes encoding GFP-labeled proteins for localization studies, or tandem affinity tagged proteins for purification of complexes, for every gene in the yeast genome. This paper describes a high-throughput pipeline for accomplishing the same goals in mammalian cells. It utilizes bacterial artificial chromosomes (BACs) because these are large enough to encompass most regulatory elements and hence support expression that closely matches endogenous genes. BAC clones are engineered in 96-well plate format by “recombineering” (in vivo homologous recombination-based methods) in E. coli. The procedure is streamlined by automation in liquid handling, making it rapid, and the methodology is anticipated to be generally applicable in diverse systems.

Johnson DS et al. Systematic evaluation of variability in ChIP-chip experiments using predefined DNA targets. Genome Research 18;2008:393–403.

Results are reported here from a multisite survey of the relative capabilities of various methods associated with the detection of protein-DNA interactions by ChIP-chip (chromatin immunoprecipitation and hybridization to tiling microarrays). Eight participating laboratories were supplied human genomic DNA with spike-ins of some 100 human sequences at various concentrations. The participants didn’t know the identity, number, or quantitative range of the spike-ins. The results indicate that differences between tiling array platforms are discernable. For example, arrays of long oligonucleotides (NimbleGen and Agilent) are more sensitive at detecting very low enrichment levels than arrays of short oligonucleotides (Affymetrix). However, variation between laboratories, protocols, and algorithms is much more important than array platform in accounting for differences in performance. The results provide a benchmark against which to measure future technical improvements.

Li X, Macarthur S, Bourgon R, Nix D, Pollard DA, Iyer VN, Hechmer A, Simirenko L, Stapleton M, Luengo Hendriks CL, Chu HC, Ogawa N, Inwood W, Sementchenko V, Beaton A, Weiszmann R, Celniker SE, Knowles DW, Gingeras T, Speed TP, Eisen MB, Biggin MD. Transcription factors bind thousands of active and inactive regions in the Drosophila blastoderm. PLoS Biology 6;2008:e27.

This is a study of the DNA-binding specificity and physiologic effects of a representative set of eukaryotic transcription factors: 6 maternal and gap transcription factors in Drosophila that initiate anterior-posterior patterning. Studies using the ChIP-chip method reveal binding to a far larger number of genomic sites (several thousand) than these factors are known to regulate. The more highly bound regions include all of the enhancers that are known to respond to these factors, or lie in the neighborhood of developmental regulators and genes showing patterned expression that represent putative cis-regulatory elements under control of these factors. Regions bound by the factors at lower levels, however, appear not to be under regulatory control by these factors. These findings indicate that not all sites that bind transcription factors can be assumed to be under their regulatory control, and that quantitative measurements of DNA–protein interactions are required to distinguish functional from nonfunctional binding.

IMAGING

Poulitaival SM, Burnum KE, Cornett DS, Caprioli RM. Solvent-free matrix dry-coating for MALDI imaging of phospholipids. Journal of the American Society for Mass Spectrometry 19;2008:882–886.

2,5-Dihydroxybenzoic acid is ground to a fine powder in a mortar and pestle and applied to dried tissue sections for MALDI imaging by filtering the powder directly onto the tissue surface through a 20-μm stainless steel sieve. Images of lipids yielded by this procedure are of comparable quality to those obtained by air brushing application of matrix solution, but the preparation procedure is simpler and more reproducible.


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