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J Biomol Tech. 2006 September; 17(4): 294–299.
PMCID: PMC2291791

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AMINO ACID SEQUENCING

Yang W-C, Mirzaei H, Liu X, Regnier FE. Enhancement of amino acid detection and quantification by electrospray ionization mass spectrometry. Analytical Chemistry 78;2006:4702–4708. [PubMed]

Mirzaei H, Regnier F. Enhancing electrospray ionization efficiency of peptides by derivatization. Analytical Chemistry 78;2006:4175–4183. [PubMed]

For the purpose of amino acid analysis, Yang et al. design reagents for derivatizing amino acids that confer several features desirable for separation by reverse-phase liquid chromatography and detection by electrospray mass spectrometry. The reagents, N-hydroxysuccinimide esters of N-alkylnicotinic acid (the alkyl group having various chain lengths), add hydrophobicity that enhances retention on reverse-phase columns well beyond the breakthrough solvent peak, and promote ionization both through the positive charge on the tertiary amine group and the additional surface activity conferred by the alkyl chain that directs the analytes to the surface layers of electrospray droplets. Reaction between these reagents and amino acids is efficient and the reagent structure allows for isotopic labeling for comparative analyses. Mirzaei and Regnier demonstrate use of the same reagents to promote electrospray detection of peptides.

DNA CHARACTERIZATION AND GENOTYPING

Blazej RG, Kumaresan P, Mathies RA. Microfabricated bioprocessor for iontegrated nanoliter-scale Sanger DNA sequencing. Proceedings of the National Academy of Sciences, U.S.A. 103;2006:7240–7245.

A microfluidic device is described for performing all three steps in Sanger sequencing: thermal cycling, purification, and electrophoresis. The device incorporates a miniaturized reaction vessel of 250 nL for the primer extension reaction to eliminate the need for sample excess, and utilizes specific affinity for an oligonucleotide complementary to a region immediately 3′ to the sequencing primer for purification of the extension products. Beginning with only 1 fmol of DNA template, the device achieves read-lengths of up to 556 consecutive bases with 99% accuracy, and has performed up to 40 runs without degradation of performance.

Salk JJ, Sanchez JA, Pierce KE, Rice JE, Soares KC, Wangh LJ. Direct amplification of single-stranded DNA for pyro-sequencing using linear-after-the-exponential (LATE)-PCR. Analytical Biochemistry 353;2006:124–132. [PubMed]

This paper addresses a bottleneck in the pyrosequencing process, now widely used for applications including SNP genotyping and allele frequency determination. Pyro-sequencing commonly involves the preparation of single-stranded templates from double-stranded PCR products, which, in many protocols, is a multistep, time-consuming process. A new method for preparing single-stranded templates is here described that involves an improved asymmetric PCR amplification procedure. By amplifying single-stranded material, this procedure circumvents the need for strand isolation and permits pyrosequencing to be performed in the same reaction vessel, thereby reducing the risk of laboratory contamination by PCR products. Existing assays can be converted to utilize this method by simple modifications to the PCR primers.

Zhou G, Kajiyama T, Gotou M, Kishimoto A, Suzuki S, Kambara H. Enzyme system for improving the detection limit in pyrosequencing. Analytical Chemistry 78;2006:4482–4489. [PubMed]

Pyrosequencing is a sequencing-by-synthesis method in which the template-directed, polymerase-catalyzed incorporation of each of the four nucleotides that can be added to the growing chain at each position is quantitated by a luciferase reaction that indirectly measures the pyrophosphate released during nucleotide addition. This paper suggests a new method of linking pyrophosphate to the luciferase reaction in which ATP is produced from pyrophosphate by pyruvate orthophosphate dikinase using AMP and phosphoenolpyruvate as substrates. This method supports sequencing reads up to 70 bases and can work with as little as 2.5 fmol of template DNA. The method is shown to work on a simple, inexpensive sequencer with sample amounts 100X lower than conventional pyrosequencers.

Neuzil P, Zhang C, Pipper J, Oh S, Zhuo L. Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes. Nucleic Acids Research 34;2006:e77. [PubMed]

A design for a real-time PCR device that conducts one cycle in 8.5 sec (40 cycles in 5 min 40 sec) is described. The cycle consists of 2 sec for denaturation and 6.5 sec for annealing/extension. The device is capable of very rapid heating (175°C/sec) and cooling (–125°C/sec) because of its low heat capacity and the low sample volume (100 nL). The sample is placed on a cover slip and encapsulated in mineral oil. The instrument presently supports just a single PCR, but more wells can in principle be integrated to form an array for high-throughput applications.

Matsumura H, Bin Nasir KH, Yoshida K, Ito A, Kahl G, Krüger DH, Terauchi R. SuperSAGE array: the direct use of 26-base-pair transcript tags in oligonucleotide arrays. Nature Methods 3;2006:469–474. [PubMed]

Methodology is described for combining the advantages of microarrays and serial analysis of gene expression (SAGE) for genome-wide expression analysis. The approach is built upon the recently improved SAGE technique called SuperSAGE ( Matsumura H et al. PNAS 100;2003:15,718–15,723 [PubMed] ), in which 26-bp tags replace the 14-bp tags of classical SAGE and the 21-bp tags of Long-SAGE for enhanced specificity in gene-to-tag annotation. The present paper demonstrates that these 26-bp tags can be used as probes in microarray analyses. The sequences of tags in a SuperSAGE library are reproduced in microarray format using NimbleGen’s photolithographic process, along with suitable mismatch controls. Such microarrays are shown to yield reproducible expression data and provide an appropriate platform for analysis of large numbers of samples, an application for which SAGE methodologies are ill-suited. The combined microarray/ SAGE methodology has the additional advantage that it permits the study of organisms for which insufficient genome sequence information is available for microarray construction.

CARBOHYDRATE AND GLYCOPROTEIN ANALYSIS

Lohse A, Martins R, Ryborg Jørgensen M, Hindsgaul O. Solid-phase oligosaccharide tagging (SPOT): Validation on glycolipid-derived structures. Angewandte Chemie International Edition 45;2006:4167–4172.

This work provides a simple method for tagging reducing carbohydrates with fluorescent or other groups to assist in subsequent chromatographic or electrophoretic analysis. The reducing sugar is captured on a solid support by reaction with benzyl hydroxylamine capture groups to form an oxime linkage. Unreacted capture groups are then capped, and the oxime is reduced to yield a species with a reactive nitrogen to which a tag containing, for example, an aryl isothiocyanate group can be added. The tagged carbohydrate is then cleaved from the solid support by treatment with LiOH, which attacks a cleavable ester linkage incorporated into the capture group. This scheme utilizes the advantages of solid-phase chemistry in promoting reaction efficiency by adding large excesses of tagging and other reagents, while minimizing losses through simple washing to remove reaction by-products.

PEPTIDE AND PROTEIN SYNTHESIS

Steiner D, Forrer P, Stumpp MT, Plückthun A. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. Nature Biotechnology 24;2006:823–831.

Phage display is a method of long standing for selecting peptides or proteins having, for example, the ability to bind a particular ligand, from among an extensive library of variants. Filamentous phages of E. coli are conventionally used, and the polypeptides to be displayed are expressed with an N-terminal signal sequence and a C-terminal extension that encodes the phage coat protein, p3. Prior to incorporation into phage particles, the protein product must be translocated to the periplasm, usually via the Sec translocation pathway. In this paper, a different signal sequence that directs proteins for translocation via a different pathway, the signal recognition particle (SRP) pathway, is shown to yield greatly enhanced display levels (700-fold greater in the case of designated ankyrin-repeat proteins). The SRP pathway operates cotranslationally, thereby overcoming inefficient translocation via the Sec pathway due to premature protein folding in the cytoplasm. It is anticipated that this new method will expand the range of proteins that can be efficiently displayed.

Love JC, Ronan JL, Grotenbreg GM, van der Veen AG, Ploegh HL. A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nature Biotechnology 24;2006:703–707.

A high-throughput method for screening hybridomas having specificity against antigens of interest is described. Spleen cells from immune mice are allowed to settle into 0.1–1.0 nL microwells (1–3 cells/well) in an array manufactured by soft lithography. The wells are 100 μm in diameter and depth. The array fits within the boundaries of a 1" × 3" microscope slide. The array is incubated for 2–4 h in contact with a solid support pre-treated to capture antibody secreted into the culture fluid in the wells during that time. The surface of the support is then tested for specific antibodies by methods typical for commercial arrays of proteins or antibodies. Cells are then recovered by micromanipulation from wells testing positive, and can be expanded. In comparison to traditional methods of cloning by serial dilution and screening by ELISA, this procedure allows identification of cells of interest before they have been overgrown by rapidly dividing sisters, and can be used to screen 100,000 individual cells in less than 12 h. In principle, the methodology is also suitable for screening Epstein-Barr virus–transformed B cells and even primary cells from peripheral blood or tissues.

METABONOMICS

Teahan O, Gamble S, Holmes E, Waxman J, Nicholson JK, Bevan C, Keun HC. Impact of analytical bias in metabonomic studies of human blood serum and plasma. Analytical Chemistry 78;2006:4307–4318. [PubMed]

This paper documents the quantitative effects of variation in protocols for sample collection, handling, and storage on the abundance of metabolites measured during metabonomic screening by NMR spectroscopy. During the production of serum, the effects of blood clotting time are found to be small, and changes in the levels of energy metabolites can be minimized by collecting samples on ice. Stability of profiles at room temperature is high. Overall, the effects of sample-processing variables are small compared to differences between individuals. Standard procedures are recommended to control bias in clinical studies that might result from the small effects observed.

MASS SPECTROMETRY

Holle A, Haase A, Kayser M, Höhndorf J. Optimizing UV laser focus profiles for improved MALDI performance. Journal of Mass Spectrometry 41;2006:705–716. [PubMed]

Many commercially available instruments for MALDI have employed nitrogen lasers at 337-nm wavelength. These lasers are inexpensive, and standard applications have been optimized based on their characteristics. However, the maximum repetition rate of nitrogen lasers is 50 Hz, and their lifetime is only 2–6 × 107 shots. These characteristics limit use for high-throughput applications such as imaging and LC-MALDI. Solid-state Nd:YAG lasers, by contrast, can achieve repetition rates of >1000 Hz and lifetimes of 109 shots, and frequency-tripled Nd:YAG lasers (355 nm) are now commonly used. However, they are sub-optimal for use with several important matrices, including sinapinic acid (for proteins) and 3-hydroxypicolinic acid (for oligonucleotides), and give poorer results with thin-layer preparations of α-cyano-4-hydroxycinnamic acid. This study demonstrates that using a spatially structured beam profile instead of the usual Gaussian beam profile allows Nd:YAG lasers to display many of the advantageous characteristics of nitrogen lasers. This modulation of beam profile is available in the Bruker “Smartbeam” laser.

Gunawardena HP, Emory JF, McLuckey SA. Phosphopeptide anion characterization via sequential charge inversion and electron-transfer dissociation. Analytical Chemistry 78;2006:3788–3793. [PubMed]

Phosphorylation imparts an elevated propensity for peptides to form negatively charged ions and a suppression of their propensity to form positively charged ions in the gas phase. However, it is frequently difficult to localize the site(s) of phosphorylation by fragmentation in the negative-ion mode. This paper demonstrates an ion/ion reaction method that converts singly charged phosphopeptide anions to doubly protonated species suitable for structural analysis by electron transfer dissociation. This charge inversion is achieved by reaction with a triply protonated reagent ion of diaminobutane core dendrimer generation 4 (DAB-g4). This represents a novel approach to the solution of a longstanding difficulty in the mass-spectrometric analysis of phosphopetides.

Cohen SL. Ozone in ambient air as a source of adventitious oxidation. A mass spectrometric study. Analytical Chemistry 78;2006:4352–4362. [PubMed]

In MALDI mass spectrometry, the extent of oxidation of methionine and tryptophan residues, and of carbon-carbon double bonds in unsaturated phospholipids, is correlated with ambient ozone levels in the air. The levels of the oxidation artifacts are highest in samples prepared during daytime hours and in the warmer months of the year, tracking with atmospheric ozone levels. The oxidation is particularly striking when samples are allowed to dry in air in the absence of MALDI matrix. Atmospheric ozone is proposed to be responsible. Oxidation is minimized when samples are allowed to dry under vacuum or in a nitrogen atmosphere.

Vivó-Truyols G, Schoenmakers PJ. Automatic selection of optimal Savitzky-Golay smoothing. Analytical Chemistry 78;2006:4598–4608. [PubMed]

A method is described for optimizing smoothing by the Savitzky-Golay algorithm that is applicable to NMR, chromatography, and mass-spectrometry data. The method automatically selects the best window size by minimizing the difference between the autocorrelation of the instrument noise and the autocorrelation of the fitting residuals (i.e., the difference between the input signal and the smoothed signal). The autocorrelation of the instrument noise is determined beforehand using blank signals.

PROTEINS—PURIFICATION AND CHARACTERIZATION

Willard FS, Siderovski DP. Covalent immobilization of histidine-tagged proteins for surface plasmon resonance. Analytical Biochemistry 353;2006:147–149. [PubMed]

The immobilization of hexahistidine-tagged proteins for surface plasmon resonance experiments is problematic. Interaction of the His-tag with Ni2+ ions on commercially available nitriloacetic acid (NTA) chips is not stable. Alternative strategies involving the use of antibodies directed against the His-tag followed by covalent attachment of captured molecules by covalent cross-linking may result in protein inactivation through exposure to cross-linking reagents. Here, the interaction between proteins captured by Ni2+ ions on NTA chips is stabilized by functional groups on the dextran surface created by pre-treatment with N-hydroxysuccinimide (NHS)/1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide hydrochloride (ECD). Immobilized proteins are not directly exposed to reactive cross-linking reagents.

Säfsten P, Klakamp SL, Drake AW, Karlsson R, Myszka DG. Screening antibody-antigen interactions in parallel using Biacore A100. Analytical Biochemistry 353;2006:181–190. [PubMed]

This study illustrates the use of a newly commercialized array-based biosensor system, the Biacore A100, for parallel processing to increase throughput. Each flow-cell has five detection spots that can be monitored simultaneously. Using this system, 386 crude hybridoma samples are screened for desired binding characteristics in a 12-h automated run.

PROTEOMICS

Nettikadan S, Radke K, Johnson J, Xu J, Lynch M, Mosher C, Henderson E. Detection and quantification of protein bio-markers from fewer than 10 cells. Molecular and Cellular Proteomics 5;2006:895–901. [PubMed]

This paper illustrates the principle of sensitivity enhancement through miniaturization in the context of antibody microarrays. Arrays with spot diameters of 0.1–1.0 μm are constructed with capture antibodies against IL-2 and PSA. The antigens are detected at pg/mL levels using a standard fluorescence microscopy system. Cellular PSA from lysates of an average of just 6 cells is demonstrated.

Piening BD, Wang P, Bangur CS, Whiteaker J, Zhang H, Feng L-C, Keane JF, Eng JK, Tang H, Prakash A, McIntosh MW, Paulovich A. Quality control metrics for LC-MS feature detection tools demonstrated on Saccharomyces cerevisiae proteomic profiles. Journal of Proteome Research 5;2006:1527–1534. [PubMed]

Dodds ED, An HJ, Hagerman PJ, Lebrilla CB. Enhancing peptide mass fingerprinting through high mass accuracy: Exclusion of non-peptide signals based on residual mass. Journal of Proteome Research 5;2006:1195–1203. [PubMed]

These papers exemplify the power of high mass accuracy, such as that achievable by Fourier transform mass spectrometry, for distinguishing peptidic from non-peptidic features in high-throughput LC-MS experiments. Peptides can manifest only a discrete subset of masses. Within any 1-Da mass window, calculated peptide monoisotopic masses are clustered in some areas but are absent from others because they are made of a limited set of elements with characteristic mass defect values. This is made the basis of feature detection algorithms that can flag mass signals occurring in areas of low probability for exclusion from an analysis. The result is greater confidence in protein identifications and decreased numbers of unassigned features of interest.

Hernandez H, Niehauser S, Boltz SA, Gawandi V, Phillips RS, Amster IJ. Mass defect labeling of cysteine for improving peptide assignment in shotgun proteomic analyses. Analytical Chemistry 78;2006:3417–3423. [PubMed]

Bruce C, Shifman MA, Miller P, Guicicek EE. Probabilistic enrichment of phosphopetides by their mass defect. Analytical Chemistry 78;2006:4374–4382. [PubMed]

These papers extend the use of the mass defect principle to select peptides with particular structural features for consideration in proteomic experiments. Hernandez et al. change the mass defect of peptides containing a cysteine residue by derivatizing with a thiol reagent, 2,4-dibromo-(2′-iodo)acetanilide, that contains heavy elements with large mass defects. Derivatized peptides are thereby moved to regions of the mass scale unoccupied by underivatized peptides, where they can readily be recognized and identified. Bruce et al. develop an algorithm for calculating the probability that a peptide is singly, doubly, or triply phosphorylated or unphosphorylated based on its mass defect. This permits putative phosphopetides within a mixture of unphosphorylated peptides to be rapidly selected for subsequent phosphorylation site mapping and sequencing based on their rank order of phosphorylation likelihood.

Stead DA, Preece A, Brown AJP. Universal metrics for quality assessment of protein identifications by mass spectrometry. Molecular and Cellular Proteomics 5;2006:1205–1211. [PubMed]

A standard statistical methodology, the receiver-operating characteristic (ROC) plot, is utilized to describe the discrimination between true matches and false matches afforded by various search metrics for peptide mass fingerprint data (e.g., hit ratio, sequence coverage, excess of fragments representing complete digestion). The ROC technique consists of plotting the fraction of true positives against the fraction of false positives for possible acceptance criteria for search results based on such metrics. The area under the curve indicates the quality of the search—a value of 1.0 indicates a perfect test in which there is no overlap between the distributions of scores for true and false positives, whereas a value of 0.5 indicates that the scores for the two groups do not differ and the test cannot discriminate true from false positives. The methodology is used to evaluate the usefulness of various metrics to discriminate true from false positives, and a combination of metrics is suggested to optimize acceptance criteria.

MICROARRAYS

Lynch JL, deSilva JS, Peeva VK, Swanson NR. Comparison of commercial probe labeling kits for microarray: Towards quality assurance and consistency of reactions. Analytical Biochemistry 355;2006:224–231. [PubMed]

Seven currently available commercial labeling kits are compared with respect to their sensitivity, reliability, and the consistency of reactions. The kits cover three probe-labeling methods—direct labeling, indirect labeling, and dendrimer technology. The kits are tested on common samples, employing manufacturers’ recommended procedures. The central result is that although all kits successfully labeled probes, differences in performance exist between different kits. In the authors’ hands, the Strata-gene Fairplay Labeling Kit is the most sensitive, the Invitrogen SuperScript Indirect cDNA Labeling System shows the most reproducible pattern of gene expression and the least technical variation, and the Promega Pronto! Plus System shows the smallest dye bias (although with greater levels of technical variation). There is also variation in the processing time, and ability to perform quality control. The results indicate the need for core laboratories to perform comparative evaluations of their own before standardizing on one labeling kit.

Siddiqui AS, Delaney AD, Schnerch A, Griffith OL, Jones SJM, Marra MA. Sequence biases in large scale gene expression profiling data. Nucleic Acids Research 34;2006:e83. [PubMed]

Using available gene expression data from one microarray method (Affymetrix GeneChip) and four sequencing methods (LongSAGE, LongSAGE-Lite, MPSS-Classic, and MPSS-Signature), variation between methods in transcripts detected and in measured level of expression are interpreted in terms of G+C content. LongSAGE has the smallest bias toward G+C-rich sequences, and MPSS the greatest bias. With Affymetrix, the extent of bias depends on the processing software employed.

Le Brigand K, Russell R, Moreilhon C, Rouillard J-M, Jost B, Amiot F, Magnone V, Bole-Feysot C, Rostagno P, Virolle V, Defamie V, Dessen P, Williams G, Lyons P, Rios G, Mari B, Gulari E, Kastner P, Gidrol X, Freeman TC, Barbry P. An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes. Nucleic Acids Research 34;2006:e87. [PubMed]

In an Anglo-French collaborative project, libraries of 50–52mer oligonucleotides have been selected as probes for human and mouse RNA expression. These probe libraries are made available on an open-source basis, and 3,500 arrays have been distributed to the academic community. Some 148,993 oligonucleotides were synthesized against human transcripts and 121,703 against mouse. From these, 25,342 human and 24,109 mouse probes have been selected for distribution. Selection has been performed on the basis of BLAST specificity, the number of ESTs matching each probe, and the distance to the 3′ end of the target mRNA.

FUNCTIONAL GENOMICS AND PROTEOMICS

Carninci P, Sandelin A, Lenhard B, Katayama S, Shimokawa K, Ponjavic J, Semple CA, Taylor MS, Engstrom PG, Frith MC, Forrest AR, Alkema WB, Tan SL, Plessy C, Kodzius R, Ravasi T, Kasukawa T, Fukuda s, Kanamori-Katayama M, Kitazume Y, Kawaji H, Kai C, Nakamura M, Konno H, Nakano K, Mottagui-Tabar S, Arner P, Chesi A, Gustincich S, Persichetti F, Suzuki H, Grimmond SM, Wells CA, Orlando V, Wahlestedt C, Liu ET, Harbers M, Kawai J, Bajic VB, Hume DA, Hayashizaki Y. Genome-wide analysis of mammalian promoter architecture and evolution. Nature Genetics 38;2006:626–635. [PubMed]

This large-scale study redefines the features of promoters in mammalian cells, and contributes to understanding the diversity, evolutionary conservation and regulation of promoters on a genome-wide scale. The data are derived primarily from cap analysis of gene expression (CAGE), which is believed to identify genuine start sites. Though analysis of 145 mouse and 41 human libraries, the location and properties of transcriptional start sites (TSS) are reported, numbering 729,504 TSS in the mouse and 665,278 in human. Of these, 159,075 in mouse and 177,563 in human occur in clusters in which two or more CAGE tags bind. The methodology supports quantitative analysis of promoter usage. In protein-coding transcription units, alternative start sites are commonly associated with the 5′ end of the full-length cDNA. Furthermore, clusters of start sites are also associated with the majority of individual exons and with 3′ UTRs. TATA boxes are associated with narrow clusters. These clusters are associated with genes that are expressed in a tissue-specific manner and that are strongly conserved between species. However, this type represents a minority of TSS. The majority of clusters are broad and have no dominant TSS. These are usually based on CpG islands. The results of this study have extensive implications for future research on tissue-specific promoters and the cis-acting elements associated with them.

Reifschneider NH, Goto S, Nakamoto H, Takahashi R, Sugawa M, Dencher NA, Krause F. Defining the mitochondrial proteomes from five rat organs in a physiologically significant context using 2D blue-native/SDS-PAGE. Journal of Proteome Research 5;2006:1117–1132. [PubMed]

Mitochondrial proteins are solubilized with digitonin (to include membrane-bound proteins) and then fractionated by blue-native gel electrophoresis under conditions that preserve protein-protein interactions. The components of complexes separated in this way are then resolved by SDS-PAGE for mass spectrometric identification. This methodology constitutes a systematic approach to protein interactome mapping.


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