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J Biomol Tech. 2004 December; 15(4): 326–331.
PMCID: PMC2291708

Article Watch

PROTEIN SEQUENCING

Zhong H, Zhang Y, Wen Z, Li L. Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis. Nature Biotechnology 22;2004:1291–1296.

This paper reports the finding that when small proteins are hydrolyzed in 6 N HCl for 1 min while exposed to microwave irradiation from a domestic microwave oven, the proteolytic fragments produced are predominantly the products of cleavage at single sites in the polypeptide chain. They contain the entire portion of the protein N-terminal to the cleavage site and the entire portion C-terminal to the site. Internal fragments produced by cleavage at more than one position in the same chain are rare. Furthermore, the rates of cleavage of different peptide bonds are relatively uniform. Given these circumstances, MALDI mass spectrometry can be used to determine extensive stretches of amino acid sequence by measuring the increments in mass of successive members of the N- or C-terminal ion series. This method is shown to yield fragments sufficient to deduce the entire sequence of lysozyme, and to identify six phosphorylation sites in α-casein. Twenty eight small proteins from an E. coli lysate are also identified. This work will be of interest to investigators seeking methods for “top-down” identification of proteins.

DNA CHARACTERIZATION AND GENOTYPING

Vincent M, Xu Y, Kong H. Helicase-dependent isothermal DNA amplification. EMBO Reports 5;2004:795–800. [PubMed]

In this proof-of-principle study, a simple isothermal DNA amplification method is used to achieve a millionfold amplification of target sequence of up to 400 bp in plasmid or genomic DNA. The method differs from polymerase chain reaction, which uses thermal denaturation to separate two DNA strands for replication, by instead employing a DNA helicase, thus mimicking cellular replication mechanisms. Proteins that bind to single-stranded DNA are also added to prevent reannealing of the template DNA. By eliminating the need for thermal cyclers, it is hoped that this methodology will become the basis for simple, portable DNA diagnostic devices for use in the field or at point-of-care.

Hart JR, Johnson MD, Barton JK. Single-nucleotide polymorphism discovery by targeted DNA photocleavage. Proceedings of the National Academy of Sciences, U.S.A. 101;2004:14040–14044.

A method for rapid SNP discovery and localization is described. SNPs are detected in genomic DNA from pooled samples. A portion of the genomic DNA selected for study is amplified by PCR, and these PCR products are denatured and annealed to produce mismatches at the sites of SNPs. The DNA is then treated with an intercalating reagent, which binds at sites of mismatch, and then catalyzes site-specific DNA cleavage upon ultraviolet irradiation. The DNA is labeled with a fluorescent tag at ends incorporated by design during the earlier PCR step, and cleaved products are detected as length differences by capillary electrophoresis. Alleles with frequency as low as 5% are detectable by this method. It is hoped that the approach will avoid the high costs associated with re-sequencing. Although not all polymorphisms are detected, false positives are avoided.

CARBOHYDRATES, GLYCOLIPIDS, AND GLYCOPROTEINS

Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC. Exporing the O-GlcNAc proteome: Direct identification of O-GlcNAc-modified proteins from the brain. Proceedings of the National Academy of Sciences, U.S.A. 101;2004:13132–13137.

The modification of serine and threonine residues by covalent attachment of β-N-acetylglucosamine (O-GlcNAc) is a dynamic, intracellular process that has been associated with nutrient sensing, protein degradation, and gene expression. Perturbations in the process have been associated with cancer, Alzheimer’s disease, and diabetes. The present work utilizes an engineered β-1,4-galactosyltransferase to tranfer a ketone-containing galactose analog selectively to the C-4 hydroxyl group of GlcNAc. The ketone group is then reacted with an aminooxy biotin nucleophile, permitting the enrichment of O-GlcNAc-modified proteins and localization of modified peptides in protein digests by avidin affinity chromatography. Twenty five O-GlcNAc-modified proteins are identified in brain, including regulatory proteins associated with gene expression, neuronal signaling, and synaptic plasticity.

MACROMOLECULAR SYNTHESIS

Clayton D, Shapovalov G, Maurer JA, Dougherty DA, Lester HA, Kochendoerfer GG. Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences, U.S.A. 101;2004:4764–4769.

The mechanosensitive bacterial ion channel protein, MscL, is made by sectional synthesis and subsequent ligation of the sections, using conditions that improve conditions for the very hydrophobic species involved. Cysteine residues introduced for the purpose of ligation are masked by reaction with sulfhydryl-reactive molecules to produce side chains similar to those in the biological sequence at the affected positions. Biotin is also incorporated for subsequent fluorescence microscopy. To reconstitute the completed protein into membranes for functional testing, it is transferred to an intermediate solution in 2,2,2-trifluoroethanol before reconstitution into vesicles. The resulting protein is functionally active in electrophysiological tests. The chemical synthesis approach permits the introduction of fluorescent unnatural amino acids and other chemical modifications to facilitate study of molecular function.

Becker CFW, Strop P, Bass RB, Hansen KC, Locher KP, Ren G, Yeager M, Rees DC, Kochendoerfer GG. Conversion of a mechanosensitive channel protein from a membrane-embedded to a water-soluble form by covalent modification with amphiphiles. Journal of Molecular Biology 343;2004:747–758. [PubMed]

Four cysteine residues incorporated into a transmembrane spanning segment of chemically synthesized MscL (see preceding review) are modified in the native protein with amphiphilic groups to render the protein soluble in water. Amphiphiles of different chain length are synthesized for this purpose. One conferred a water-solubility as high as 4 mg/mL in the absence of detergent. The modified protein is shown to exist in native-like α-helical pentamers. It is hoped that attachment of amphiphiles in this way will aid in crystallization, solution NMR, and electron microscopic studies of intrinsic membrane proteins.

Xiong A-S, Yao Q-H, Peng R-H, Li X, Fan H-Q, Chen Z-M, Li Y. A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences. Nucleic Acids Research 32;2004:e98. [PubMed]

Genes of 1.0–5.4 kb are synthesized using a two-step procedure. In the first step, 10–12 60-mer oligonucleotides with 20 bp overlaps are chemically synthesized. These are mixed together, and PCR performed using polymerase Pfu to produce DNA pieces of 500 bp. In the second step, 5–10 of these PCR products are mixed and employed as template for a second PCR using the two outermost 60-mer oligonucleotides as primers and pyrobest DNA polymerase. The method takes as little as 5–7 days, and is suitable for genes with high G + C content, repetitive sequences, or complex secondary structure.

Richmond KE, Li M-H, Rodesch MJ, Patel M, Lowe AM, Kim C, Chu LL, Venkataramaian N, Flickinger SF, Kaysen J, Belshaw PJ, Sussman MR, Cerrina F. Amplification and assembly of chip-eluted DNA (AACED): A method for high-throughput gene synthesis. Nucleic acids Research 32;2004:5011–5018. [PubMed]

Zhou X, Cai S, Hong A, You Q, Yu P, Sheng N, Srivannavit O, Muranjan S, Rouillard JM, Xia Y, Zhang X, Xiang Q, Ganesh R, Zhu Q, Matejko A, Gulari E, Gao X. Microfluidic picoarray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences. Nucleic Acids Research 32;2004:5409–5417. [PubMed]

Two groups here describe studies to demonstrate the feasibility of synthesizing genes from 40–60-mer oligonucleotides made by maskless photolithography in array synthesizers. Richmond et al. use a chemically derivatized glass slide as substrate, whereas Zhou et al. use a microfluidic device in which array features are contained within microreaction chambers linked together via fluid microchannels. In both cases, synthesis chemistry originally implemented to make arrays for RNA expression analysis required optimization to improve yields of full-length oligonucleotides needed for the more demanding preparative application. Elution of oligonucleotides from the chips, ligation, and fusion PCR are then used to make and amplify DNA constructs of up to 10 kb.

MASS SPECTROMETRY

Trauger SA, Go EP, Shen Z, Apon JV, Compton BJ, Bouvier ESP, Finn MG, Siuzdak G. High sensitivity and analyte capture with desorption/ionization mass spectrometry on silylated porous silicon. Analytical Chemistry 76;2004:4484–4489. [PubMed]

DIOS (desorption/ionization on silicon), a technique that uses a porous silicon surface to produce ions, is especially well suited for analysis of small molecules because it does not use a matrix. Here, the technique is extended by silylation of the oxidized porous silicon surface with commercially available silylating agents. The resulting surfaces are resistant to air oxidation and acid/base hydrolysis, and permit analyte adsorption to the surface via hydrophobic interactions, leaving in solution salts and other nonvolatile substances that can interfere with ionization. The surface thus acts as a solid-phase extraction device that enables sample cleanup simply by removing the liquid phase after adsorption. The methodology is employed to analyze peptides in protein digests, metabolites in biological fluids, and inhibitors in complex with cognate enzymes. A detection limit of 800 ymol is reported for des-Arg9-bradykinin on a pentafluorophenyl-functionalized DIOS chip.

Low W, Kang J, DiGruccio M, Kirby D, Perrin M, Fischer WH. MALDI-MS analysis of peptides modified with photolabile arylazido groups. Journal of the American Society of Mass Spectrometry 15;2004:1156–1160.

This paper addresses the general question of how to use MALDI to detect the intact molecular ions of peptides that are light sensitive. An ultraviolet laser is used to detect peptide derivatives containing the photolabile p-azidobenzoyl group commonly employed for characterizing molecular interactions. With α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid as MALDI matrix, photo-induced degradation products dominate the spectra, but with sinapinic acid the intact molecular ion predominates. This observation is explained by the strong absorbance of the matrix at the laser wavelength, thus providing protection of the analyte.

Han J, Schey KL. Proteolysis and mass spectrometric analysis of an integral membrane: Aquaporin 0. Journal of Proteome Research 3;2004:807–812. [PubMed]

Detergent- and CNBr-free methodology for sequence analysis of an intrinsic membrane protein is described. Digestion with trypsin is accomplished in bicarbonate buffer by adding acetonitrile to a concentration optimized empirically. Full sequence coverage is observed both with MALDI and electrospray ionization. Conditions for digestion with a second enzyme are also described, since not all proteins are anticipated to yield tryptic peptides that behave as favorably. Pepsin digestion in 10% formic acid (pH 1.2) is observed to give peptides of mass 500–3000 Da, and, again, complete sequence coverage.

PROTEINS—PURIFICATION AND CHARACTERIZATION

Doneanu CE, Gafken PR, Bennett SE, Barofsky DF. Mass spectrometry of UV-cross-linked protein-nucleic acid complexes: Identification of amino acid residues in the single-stranded DNA-binding domain of human replication protein A. Analytical Chemistry 76;2004:5667–5676. [PubMed]

The human replication protein A is photochemically cross-linked to the oligonucleotide dT30 to form “zero-length” covalent bonds at sites of contact. Nucleoprotein complexes with 1:1 stoichiometry are isolated by SDS-PAGE and digested with trypsin. Three cross-linked tryptic peptides are recovered. Their identities are established by a combination of tandem mass spectrometry and digestion with carboxypeptidase Y. IMAC ZipTips (Millipore) are used to purify and concentrate the carboxypeptidase-digested peptides for MALDI mass spectrometry. The results permit identification of three phenylalanine residues in the protein that make contact with the DNA, presumably by base-stacking interactions.

Geyer H, Geyer R, Pingoud V. A novel strategy for identification of protein-DNA contacts by photocrosslinking and mass spectrometry. Nucleic Acids Research 32;2004:e132. [PubMed]

In this study, the restriction enzyme MboI is photochemically cross-linked to 12 bp double-stranded DNA containing the MboI recognition sequence. The photoactivatable residue, 5-iododeoxyuridine is incorporated into the DNA at the T in the recognition sequence GATC because the substitution at that position causes the least change to binding affinity. Protease digestion of the cross-linked protein pool is followed by isolation of nucleopeptides by IMAC affinity chromatography. The DNA is then removed by hydrolysis with 45% hydrogen fluoride before mass spectral identification of the peptides. Use of various proteases results in assignment of the cross-link to a single lysine residue, which becomes modified with deoxyuridine detectable in the mass spectra.

Tjernbeerg A, Carnö S, Oliv F, Benkestock K, Edlund P-O, Griffiths WJ, Hallén D. Determination of dissociation constants for protein–ligand complexes by electrospray ionization mass spectrometry. Analytical Chemistry 76;2004:4325–4331. [PubMed]

Dissociation constants for the binding of low molecular weight ligands to proteins are measured by a mass spectrometric procedure that avoids making the assumption that the relative mass spectral signal strengths of bound and unbound protein in the gas phase reflect the relative concentrations of bound and unbound protein in solution. Independence of this assumption is made possible by the introducing a mass spectral response factor, which reflects the mass spectral signal strength of a protein that is ligand-saturated in solution relative to the unbound protein. In this way, complexes that dissociate easily in the gas phase (usually those stabilized by hydrophobic forces) can be studied as well as complexes that are relatively stable in the gas phase (usually those stabilized by polar interactions). Both direct binding and competition assays making use of this principle are described.

Markova N, Hallén D. The development of a continuous isothermal titration calorimetric method for equilibrium studies. Analytical Biochemistry 331;2004:77–88. [PubMed]

Isothermal titration calorimetry has proved to be a valuable method for characterizing molecular interactions of proteins. In this report, a method for continuous slow injection of titrant into the calorimetric vessel is documented. The method shortens the time needed to perform an experiment by a factor of 10, and increases the number of data points collected 30-fold compared with classical isothermal titration calorimetry. The increased data density permits higher precision in estimating thermodynamic parameters, and supports improved discrimination between different binding models. The range of affinities that can be measured is also expanded by three orders of magnitude, making it possible to determine equilibrium dissociation constants as low as 10 pM.

Ferracci G, Seagar M, Joël C, Miquelis R, Lévêque C. Real time analysis of intact organelles using surface plasmon resonance. Analytical Biochemistry 334;2004:367–375. [PubMed]

Synaptic vesicles in rat brain homogenates are immobilized on Biacore chips via antibodies to synaptophysin or synaptotagmin, and then used to measure interactions of proteins with the vesicle by surface plasmon resonance. The vesicle chips are stable for days and allow repetitive use with multiple analytes. This methodology provides a way to study interactions of organelle-bound proteins in their native environment.

PROTEOMICS

Li X-M, Patel BB, Blagoi EL, Patterson MD, Seeholzer SH, Zhang T, Damle S, Gao Z, Boman B, Yeung AT. Analyzing alkaline proteins in human colon crypt proteome. Journal of Proteome Research 3;2004:821–833. [PubMed]

Proteins with pI higher than 8 are estimated to include about 40% of the human proteome, and include proteins with important cellular functions, such as ribosomal proteins, mitochondrial proteins, histones, and other DNA-binding proteins. Yet basic proteins remain challenging to analyze by 2-D gel electrophoresis. In this study of basic proteins from colonic crypts, improvements in resolution are documented for cup-loading the sample at the anode, and inclusion of 1.2% hydroxyethyl disulfide (DeStreak), 15% 2-propanol, and 5% glycerol in the rehydration buffer. Deployment of these precautions results in well-resolved spots up to pH 11.

Wu CC, MacCoss MJ, Howell KE, Matthews DE, Yates JR III. Metabolic labeling of mammalian organisms with stable isotopes for quantitative proteomic analysis. Analytical Chemistry 76;2004:4951–4959. [PubMed]

A whole rat is raised for 44 days on a diet enriched in 15N to provide metabolically labeled tissue proteins for use as internal standards for proteomic experiments. The diet consists of protein-free rodent food supplemented with 15N-labeled algal cells in powdered form. This regimen is found to have no apparent deleterious effect on health, despite low weight gain due to a restricted feeding schedule. The average 15N enrichment of amino acids ranges from 74% in brain to 92% in plasma. The 15N-labeled protein from liver are used as an internal standard to compare the changes in levels of 310 individual proteins in the livers of rats treated for 4 h with cycloheximide to inhibit protein synthesis. Using a shotgun proteomic method, proteins from both treated and control rats are compared with the same 15N-labeled internal standard mixture to increase sensitivity of the test procedure for detection of changes in expression levels. Although the measured factor changes are modest (maximum 2.6), the method may be used to study the whole body effects of a wide variety of treatments.

Kutz KK, Schmidt JJ, Li L. In situ tissue analysis of neuropeptides by MALDI FTMS in-cell accumulation. Analytical Chemistry 76;2004:5630–5640. [PubMed]

Neuropeptides from neuroendocrine organs of the Jonah crab are obtained by direct MALDI of tissue fragments prepared by washing in acidified methanol and impregnating with 2,5-dihydroxybenzoic acid matrix. The peptide ions are analyzed in a Fourier transform mass spectrometer. Sensitivity of detection of low abundance peptides is enhanced by an in-cell ion accumulation procedure that permits the accumulation of ions from multiple shots of the laser without accumulating noise. De novo sequencing of peptides is then accomplished by sustained off-resonance irradiation and collision-induced dissociation. Two additional innovations are improvement in mass accuracy to 2 ppm by using in-cell ion accumulation to introduce calibrant ions into the analysis cell from a separate calibrant spot on the target; and employment of a pulse sequence that permits coverage of a wide mass range (m/z 215–9000) by centering quadrupole collection of ions at different masses for successive laser shots.

MICROARRAYS

Calin GA, Liu C-G, Sevignani C, et al. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proceedings of the National Academy of Sciences, U.S.A. 101;2004:11755–11760.

A microarray of microRNA (miRNA) probes is used to compare miRNA expression in human B cell chronic lymphocytic leukemia cells and in normal CD5+ B cells. The array contains 40-mer oligonucleotides probes for 161 human and 84 mouse precursor miRNAs. In some cases, probes for both the active sequence and the precursor are represented, allowing simultaneous expression analysis of the mature miRNA and its precursor. Significant differences in miRNA expression pattern between chronic lymphocytic leukemia cells and normal cells are observed, suggesting that miRNA expression may play a role in the pathogenesis of human cancer.

Weil MR, Widlak P, Minna JD, Garner HR. Global survey of chromatin accessibility using DNA microarrays. Genome Research 14;2004:1374–1381. [PubMed]

Chromatin condensation/relaxation is believed to mediate the availability of specific DNA sequences to regulatory proteins and hence to play a major role in the regulation of transcription. In this study, DNA is fractionated according to its state of accessibility and then applied to commercial comparative genomic hybridization (CGH) microarrays to provide high resolution, high throughput assessment of chromatin accessibility at the level of individual genes. Chromatin is fractionated according to its condensation state either by differential solubility of mono- and oligo-nucleosomes in specific buffers, or by controlled Dnase 1 digestion and selection of the large refractory fragments representing condensed chromatin. Study of the breast tumor cell line, MCF7, shows that the two methods for selecting DNA of different accessibility correlate well. It is hoped that data acquired using this methodology will provide opportunities to identify accessibility signatures for functionally related genes, and to assign tissue- and disease-specific components to these signatures.

BIOINFORMATICS

Fernandez-Escamilla A-M, Rousseau F, Schymkowitz J, Serrano L. Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins. Nature Biotechnology 22;2004:1302–1306.

This paper presents a statistical mechanics algorithm, TANGO, that assigns the relative propensity of sequences of peptides or of different sections of polypeptide within a protein, or of different mutant sequences of a single protein, to form buried, intermolecular β-sheets, as opposed to other competing conformations, such as β-helix, β-sheet, β-turn. Intermolecular β-sheet formation is a common mechanism for protein aggregation. The algorithm distinguishes this β-aggregation propensity from β-sheet propensity. The aggregation behavior of 179 peptides from 21 different proteins in the literature, and of 71 peptides from disease-related proteins studied by the authors, is predicted. The algorithm also predicts pathogenic and protective mutations in the Alzheimer β-peptide, human lysozyme, and transthyretin. The algorithm provides an opportunity to improve the aggregation properties of proteins for laboratory study and for pharmaceutical production. It is available at http://tango.embl.de/.

FitzGerald PC, Shlyakhtenko A, Mir AA, Vinson C. Clustering of DNA sequences in human promoters. Genome Research 14;2004:1562–1574. [PubMed]

The enduring question of which DNA sequences in regions bound by transcription factors are biologically relevant is addressed here by searching for DNA sequences that localize near transcription start sites (TSS). The study utilizes new information about the location of genes in the complete human genome, and the experimental definition of large numbers of TSS, to define regions in which to search. The distribution of 8-mer sequences in 13,010 human promoter regions from −2500 to 500 bp relative to the TSS is described. Nine consensus sequences are defined, eight of which cluster between −100 and the TSS. Seven of these are known binding sites for transcription factors, but one, Clus1, is not a known transcription factor binding site. The remaining cluster is a Kozak sequence that localizes downstream of the TSS. When combined with mRNA expression data, these results indicate an association of three of the sequences, ETS, NRF-1, and Clus1, and with housekeeping genes, while TATA is more frequently associated with tissue-specific genes. This work exemplifies the complexity of defining weak consensus sequences in extensive regions of DNA.


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