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Wolfsberg TG, Wetterstrand KA, Guyer MS, Collins FS, Baxevanis AD. A user’s guide to the human genome. Nature Genetics 35;2003:Supplement pp1–79.
This guide is described as “a peer reviewed how-to manual [that] guides the reader through some of the basic tasks facing anyone whose work might be facilitated by an improved understanding of the online resources that make sense of annotated genomes.” It provides a series of worked examples illustrating the types of data available, how the data may be browsed, and instructions for using commonly employed tools for sequence-based discovery.
He S, Bauman D, Davis JS, Loyola A, Nishioka K, Gronlund JL, Reinberg D, Meng F, Kelleher N, McCafferty DG. Facile synthesis of site-specifically acetylated and methylated histone proteins:Reagents for evaluation of the histone code hypothesis. Proceedings of the National Academy of Sciences, U.S.A. 100;2003:12033–12038.
Histones modulate gene expression and chromatin assembly/remodeling through complex, combinatorial modifications of their N-terminal sequences involving methylation, acetylation, and phosphorylation. To deconvolute this so-called “histone code,” synthetic histones with specific combinations of modifications are required. This paper describes a strategy for their production involving chemical synthesis of the modified N-terminal segment by tBoc chemistry, followed by ligation to a recombinantly produced C-terminal segment engineered to contain an N-terminal cysteine residue for use in the ligation chemistry.
Chin JW, Cropp TA, Anderson JC, Mukherji M, Zhang Z, Schultz PG. An expanded eukaryotic genetic code. Science 301;2003:964–967. [PubMed]
Yeast strains are engineered to contain tRNA and aminoacyl-tRNA synthetase pairs appropriate for high efficiency, high fidelity incorporation of unnatural amino acids in response to the nonsense TAG codon. Five amino acids are incorporated: p-acetyl-L-phenylalanine (in which the keto group allows selective modification of proteins with an array of hydrazine- and hydroxylamine-containing reagents in vivo and in vitro; p-benzoyl-L-phenylalanine and p-azido-L-phenylalanine (that can be used for photocrosslinking in vivo and in vitro; p-iodo-L-phenylalanine (in which the heavy atom may be useful for phasing X-ray structure data); and O-methyl-L-tyrosine (in which the methyl group can be substituted with an isotopically labeled group for NMR structural experiments.
Traverso G, Diehl F, Hurst R, Shuber A, Whitney D, Johnson C, Levin B, Kinzler KW, Vogelstein B. Multicolor in vitro translation. Nature Biotechnology 21;2003:1093–1097.
Lysine derivatized with any of four fluorescent dyes, BODIPY-FL, BODIPY-TMR, BODIPY-TR, and BODIPY-650/655 is shown to be incorporated into proteins during in vitro translation. This is useful for multiplexing analyses of in vitro-translated proteins from related sources. In this report, DNA from the stools of individuals at risk for colorectal cancer is amplified by PCR, and DNA from the APC gene is selected, since mutation in this gene initiates the majority of colorectal tumors. This DNA is aliquoted to give pools containing small numbers of molecules. In vitro transcription and translation of the genes in each pool is then performed with incorporation of fluorescent tags and antigenic markers into the synthesized protein. Differently tagged proteins are then pooled for multiplexed analysis by SDS-PAGE to detect truncated protein species that are commonly produced by mutations in the APC gene.
Lohmann KK, von der Lieth C-W. GLYCO-FRAGMENT: A web tool to support the interpretation of mass spectra of complex carbohydrates. Proteomics 3;2003:2028–2035. [PubMed]
A web-based routine, GLYCO-FRAGMENT, is described. This tool computes the mass values for fragment ions that may be derived from defined glycan structures. The output is intended to facilitate manual assignment of peaks found in the mass spectra of complex carbohydrates. The software is accessible at http://www.dkfz.de/spec/projekte/fragments/.
Mechref Y, Novotny MV. Structural characterization of oligosaccharides using MALDI-TOF/TOF tandem mass spectrometry. Analytical Chemistry 75;2003:4895–4903. [PubMed]
The characteristics of the Model 4700 TOF/TOF Proteomics Analyzer from Applied Biosystems are evaluated for the structural characterization of oligosaccharides and N-glycans from glycoproteins. Extensive cross-ring fragmentation provides information about linkages between monosaccharide residues. A-type ions permit α(1-4)- and α(1-6)-linked isobaric structures to be clearly distinguished. Abundant A- and X-type ions allow determination of linkages in N-glycans. The molar ratios of isomeric glycans in mixtures can be determined from MS/MS spectra by observation of relative signal strengths.
Kennedy GC, Matsuzaki H, Dong S, Liu W, Huang J, Liu G, Su X, Cao M, Chen W, Zhang J, Liu W, Yang G, Di X, Ryder T, He Z, Surti U, Phillips MS, Boyce-Jacino MT, Fodor SPA, Jones KW. Large-scale genotyping of complex DNA. Nature Biotechnology 21;2003:1233–1237.
The use of an oligonucleotide microarray for the simultaneous genotyping of 14,548 human single nucleotide polymorphisms (SNPs) is documented. The methodology works without the need to use locus-specific primers or automation. The target DNA is digested with a restriction enzyme, ligated with an adaptor, and then amplified with a common primer. The complexity (number of base pairs) of genomic target DNA being sampled is limited to minimize cross-hybridization and nonspecific signals. This is done by selecting fragments of 400–800 bp in a process that employs amplification conditions optimized for that size range. Greater than 99% accuracy is achieved.
Ivnitski D, O’Neil DJ, Gattuso A, Schlicht R, Calidonna M, Fisher R. Nucleic acid approaches for detection and identification of biological warfare and infectious agents. BioTechniques 35;2003:862–869. [PubMed]
Polymerase chain reaction–based technologies for detection of biological warfare agents are reviewed. These include quantitative (real-time) PCR, the use of microfluidic devices, and the use of DNA microarrays. In accompanying articles (Peruski LF, Peruski AH. BioTechniques 35;2003:840–846 [PubMed]; and Andreotti PE, Ludwig GV, Peruski AH, Tuite JJ, Morse SS, Peruski LF. BioTechniques 35;2003:850–859 [PubMed]) DNA approaches are compared to immunochemical methods with regard to sensitivity, specificity, speed, portability, and required operator skills. Methodological improvements inspired by fears of biological warfare will hopefully prove beneficial within the wider context of public health for control of naturally spread infectious agents.
Pieper R, Gatlin CL, Makusky AJ, Russo PS, Schatz CR, Miller SS, Su Q, McGarth AM, Estock MA, Parmar PP, Zhao M, Huang S-T, Zhou J, Wang F, Esquer-Blasco R, Leigh Anderson N, Taylor J, Steiner S. The human serum proteome: Display of nearly 3700 chromatographically separate protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Proteomics 3,2003:1345–1364. [PubMed]
This extensive analysis of serum proteins addresses the large dynamic range of protein concentrations by removal of the nine most abundant serum proteins by use of a mixed-bed polyclonal antibody affinity column, serial anion exchange and size exclusion chromatography, and finally 2D electrophoresis of the fractions. The number of proteins identified, 325, may be compared with 490 proteins identified in the study of Adkins JN, et al. (Molecular and Cellular Proteomics 1;2002:947–955) [PubMed], in which 2D liquid chromatography of tryptic peptides was the methodology employed. Both approaches achieve a detection sensitivity of 1 ng protein per milliliter of serum. However, the 2-D gel method discriminates many examples of protein isoforms produced by post-translational modification. Among the large number of low abundance proteins of intracellular origin that are presumably present in serum as a result of cell leakage, few are common to both studies. The origin of the extensive differences is unknown, but could include differences in the protein content of the donor sera, varying serum preparation procedures, differences in protein fraction methods, and differences in MS analysis methods.
Peng J, Schwartz D, Elias JE, Thoreen CC, Cheng D, Marsischky G, Roelofs J, Finley D, Gygi SP. A proteomics approach to understanding protein ubiquitination. Nature Biotechnology 21;2003:921–926.
This paper presents a global screen in yeast for a post-translational modification that presents special analytical challenges because of the size of the modifying moiety (8 kDa) and the low abundance of the modified proteins, many of which are targeted for intracellular degradation in response to the modification under study. The method involves the use of Ni affinity chromatography to select ubiquitinated proteins in cells expressing 6xHis-tagged ubiquitin. The proteins that bind are digested with trypsin and the peptides are analyzed by LC/LC/MS/MS. Trypsin digestion leaves a Gly-Gly dipeptide from ubiquitin isopeptide linked to a Lys residue at the site of modification. This is detected in database searches by selecting the addition of 114.1 Da as a variable Lys modification. One hundred ten sites of ubiquitination are identified in 72 ubiquitin–protein conjugates, including 7 sites of modification on ubiquitin itself that are used to assemble multiubiquitin chains.
Troyansaya OG, Dolinski K, Owen AB, Altman RB, Botstein D. A Bayesian framework for combining heterogeneous data sources for gene function prediction (in Saccharomyces cerevisiae). Proceedings of the National Academy of Sciences, U.S.A. 100;2003:8348–8353.
Jansen R, Yu H, Greenbaum D, Kluger Y, Krogan NJ, Chung S, Emili A, Snyder M, Greenblatt JF, Gerstein M. A Bayesian networks approach for predicting protein–protein interactions from genomic data. Science 302;2003:449–453. [PubMed]
Both these papers describe the application of formal Bayesian reasoning to the integration of the disparate sources of information about protein interactions, e.g., two-hybrid screens, mRNA co-expression, subcellular co-localization, genetic linkage, and co-immunoprecipitation. Individual sources of information may be only weakly predictive of functional interaction. Each source of evidence is assessed by comparing its results against suitable examples of known interacting proteins and known noninteracting proteins. The different sources are then combined according to their reliability to give predictions with improved accuracy.
Cavanagh J, Benson LM, Thompson R, Naylor S. In-line desalting mass spectrometry for the study of noncovalent biological complexes. Analytical Chemistry 75;2003:3281–3286. [PubMed]
An arrangement for on-line desalting of proteins and protein complexes for electrospray mass spectrometry is described. Desalting is performed by size exclusion chromatography in a 760-μm ID P-6 column packed in PEEK tubing. Salts are diverted to waste with a six-port valve.
Aquilina JA, Benesch JLP, Bateman OA, Slingsby C, Robinson CV. Polydispersity of a mammalian chaperone: Mass spectrometry reveals the population of oligomers in αB-crystallin. Proceedings of the National Academy of Sciences, U.S.A. 100;2003:10611–10616.
The mammalian heat-shock protein, αB-crystallin, forms oligomeric species with a broad range of sizes. Electrospray mass spectra of the protein are therefore complicated by the presence of overlapping charge-state series from many oligomeric forms. Collision-induced dissociation is used here to strip monomeric units and their associated positive charges from the clusters, producing residual cluster ions with relatively high m/z, the signals from which can be interpreted in terms of the number of monomeric units contained and the relative abundance of different oligomeric species present. In this way, a distribution of oligomers predominantly composed of 24- to 33-mers is demonstrated, with 28-mers the most abundant. However, clusters as small as 10-mers and as large as 40-mers are also observed at low levels.
Nesvizhskii AI, Keller A, Kolker E, Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry. Analytical Chemistry 75;2003:4646–4658. [PubMed]
This paper describes statistical methodology for estimating the probability that a given protein is present in a mixture on the basis of evidence obtained by proteolytic digestion of the mixture followed by assignment of one or more peptide MS/MS spectra to that protein during database searching. The model takes into account criteria used in manual screening of MS/MS data sets, including the probability that the peptide assignments themselves are correct, the number of spectra assigned to the same or different portions of the protein’s sequence, and the existence of the same peptide sequence in more than one entry in the protein database. The method is automated, fast, and does not rely on manual validation. It provides a vehicle for standardizing the interpretation and publication of large data sets. The software to implement the method is being provided at http://systemsbiology.org/research/software.html.
Soga T, Ohashi Y, Ueno Y, Naraoka H, Tomita M, Nishioka T. Quantitative metabolome analysis using capillary electrophoresis mass spectrometry. Journal of Proteome Research 2;2003:488–494. [PubMed]
Capillary electrophoresis (CE)/MS is used to screen 1692 metabolites in Bacillus subtilis for changes during sporulation. Separate CE/MS methods are described for anionic and cationic metabolites. The mass range for individual runs is limited to 30 m/z to increase sensitivity, but a total range of 70–1027 m/z is scanned by performing multiple runs. A total of 150 of the metabolites are identified. The number of carbon atoms is estimated using 13C contributions, and charge, electrophoretic mobility, and isotopic contributions are used to select the correct structure from the LIGAND database.
Lucito R, Healy J, Alexander J, Reiner A, Esposito D, Chi M, Rodgers L, Brady A, Sebat J, Troge J, West JA, Rostan S, Nguyen KCQ, Powers S, Ye KQ, Olshen A, Venkatraman E, Norton L, Wigler M. Represenational oligonucleotide microarray analysis: A high-resolution method to detect genome copy number variation. Genome Research 13;2003:2291–2305. [PubMed]
Variations in DNA copy number in normal and cancer cells is detected using oligonucleotide microarrays in which 70-mer probes are synthesized in situ by digital optical chemistry at a density of 85,000 features per slide. Genomic DNA is prepared for hybridization by digesting with BglII, ligating adaptor sequences to the fragment ends and selectively amplifying small fragments by PCR. The DNA pool is further reduced in complexity by eliminating fragments with internal EcoRI sites. Probes are designed and empirically validated, giving an average resolution of one probe every 30 kb of genomic DNA. Amplifications, and large and small homozygous and hemizygous deletions, are detected in cancer cells. In normal human genomes, both duplications and large deletions of 100 kb to 1 Mb are also observed.
Zhang L, Miles MF, Aldape KD. A model of molecular interactions on short oligonucleotide microarrays. Nature Biotechnology 21;2003:818–821.
It is estimated that approximately 30% of probes in arrays of short oligonucleotides yield negative results. This paper contributes to improving the design of oligonucleotide microarrays by formulating and validating a free energy model for the formation of RNA–DNA duplexes on a solid support. The model is based on the nearest neighbor model. It incorporates weighting factors at each position in the probe to reflect different contributions to stability by different parts of the probe, and discriminates gene-specific and nonspecific binding. The model provides a practical guide for microarray design in terms of probe selection, and offers a means for checking data quality. A program, PerfectMatch, for data analysis using the model is available at http://bioinformatics.mdanderson.org.
Kumar R, Conklin DS, Mittal V. High-throughput selection of effective RNAi probes for gene silencing. Genome Research 13;2003:2333–2340. [PubMed]
Mousses S, Caplen NJ, Cornelison R, Weaver D, Basik M, Hautaniemi S, Elkahloun AG, Lotufo RA, Choudary A, Dougherty ER, Suh E, Kallioniemi O. RNAi microarray analysis in cultured mammalian cells. Genome Research 13;2003:2341–2347. [PubMed]
Two groups report the use of microarray-based methods for highly parallel quantitative screening of siRNAs to test efficacy in silencing target genes. siRNAs are arrayed on a glass slide in a transfection matrix, and the slide is then covered with a layer of adherent cells. Only cells growing in close proximity to a DNA spot are transfected, producing groups of transfected cells within an untransfected lawn. The method is validated by monitoring reduction in the levels of expression of a cognate target gene fused to green fluorescent protein as a reporter. This method is designed to assist in the design of large-scale siRNA libraries.
Noy D, Calhoun JR, Lear JD. Direct analysis of protein sedimentation equilibrium in detergent solutions without density matching. Analytical Biochemistry 320;2003:185–192. [PubMed]
Use of analytical ultracentrifugation for characterizing membrane proteins solubilized in detergent is complicated by alterations to the molecular weight of the protein that arise through the binding of detergent molecules. This problem may be overcome by matching the density of the detergent with the density of the solvent by adding D2O and H2O in the appropriate ratio. In cases where a detergent that cannot be densitymatched is employed, linear extrapolation of the protein’s behavior in a series of H2O/ D2O mixtures to the point of neutral detergent buoyancy is possible. The present paper describes an alternative, global nonlinear fitting method for this purpose. Use of this method enables a broader range of detergents to be used, and provides accurate information about the number of detergent molecules bound to the protein.
Haydon CE, Eyers PA, Aveline-Wolf LD, Resing KA, Maller JL, Ahn NG. Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography. Molecular and Cellular Proteomics 2;2003:1055–1067. [PubMed]
This study of a multiply phosphorylated protein incorporates a systematic, quantitative analysis of the binding and recovery of peptides to IMAC. Binding and recovery of phosphorylated and unphosphorylated peptides are reported for two different IMAC resins, the efficacy of methyl esterification is investigated, and various binding and elution conditions are evaluated. Although the IMAC technique is concluded to be feasible for identifying phosphorylation sites, the incomplete recovery of phosphopeptides observed even under optimal conditions is such as to make its use problematic in cases involving low abundance proteins and low stoichiometry of modification.
Astorga-Wells J, Swerdlow H. Fluidic preconcentrator device for capillary electrophoresis of proteins. Analytical Chemistry 75;2003:5207–5212. [PubMed]
Astorga-Wells J, Jörnvall H, Bergman T. A microfluidic electrocapture device in sample preparation for protein analysis by MALDI mass spectrometry. Analytical Chemistry 75;2003:5213–5219. [PubMed]
A device for concentration of proteins and peptides on a micro scale is described. A 1.6–2.6 cm length of PEEK tubing is flanked with segments of Nafion tubing. Nafion is negatively charged and permeable only to small cations. The Nafion segments are immersed in anode and cathode electrode chambers. Sample ions in solution initially fill the tubing assembly uniformly, but become concentrated at the anodal end by a stacking mechanism, and are retained within the assembly by their exclusion from passage through the walls of the Nafion tubing and by a pumped cathodic counterflow. Proteins concentrated in the device may then be introduced into a capillary electrophoresis system by hydrodynamic injection. Alternatively, proteins/peptides may be concentrated and desalted in the device and then hydrodynamically ejected onto a MALDI target plate.
Martin K, Hart C, Liu J, Leung W-Y, Patton WF. Simultaneous trichromic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates. Proteomics 3;2003:1215–1227. [PubMed]
A method is reported for visualizing the entire profile of proteins electroblotted to a PVDF membrane, while localizing two different, specifically targeted proteins on the same electroblot. This is achieved by first covalently coupling a fluorescent dye to the proteins, then incubating the blot with two specific reporter molecules, one conjugated to alkaline phosphatase and the other to horseradish peroxidase. The sites at which the enzymes become localized are revealed by finally incubating the blot with fluorogenic substrates appropriate for each. The three fluorophores have excitation/emission frequencies that may be detected with a laser-based scanner, a xenon-arc source with appropriate filters, or a UV epi-illuminator with photographic film or CCD camera.