PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jbtJBT IndexAssociation Homepage
 
J Biomol Tech. 2007 July; 18(3): 178–182.
PMCID: PMC2062546

ARTICLE WATCH

Abstract

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.

DNA CHARACTERIZATION AND GENOTYPING

Peters BA, St Croix B, Sjöblom T, Cummins JM, Silliman N, Ptak J, Saha S, Kinzler KW, Hatzis C, Velculescu VE. Large-scale identification of novel transcripts in the human genome. Genome Research. 2007;17:287–292. [PubMed]

LongSAGE was used to perform expression analysis on a genome-wide scale to quantitate levels of transcripts regardless of whether they correspond to known genes. Of the 660,357 transcripts analyzed from human brain mRNA, 17,409 correspond to known genes, but more than 15,000 represent transcripts from unannotated intergenic regions. Most of these transcripts are differentially expressed in various tissue types and are spliced, suggesting that they too represent bona fide genes. However, they differ in various ways from previously annotated genes. For example, three ab initio gene-detection programs detect fewer than 20% of them, and the average GC content is significantly lower than that of annotated genes. Both these features are shared with protein non-coding genes. The results of the study suggest that the human genome might contain approximately twice the number of transcribed regions that are currently annotated.

Kittler R, Surendranath V, Heninger A-K, Slabicki M, Theis M, Putz G, Franke K, Caldarelli A, Grabner H, Kozak K, Wagner J, Rees E, Korn B, Freznel C, Sachse C, Sönnichsen B, Guo J, Schelter J, Burchard J, Linsley PS, Jackson AL, Habermann B, Buchholz F. Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies. Nature Methods. 2007;4:337–344. [PubMed]

The most common approaches for generating short interfering RNAs (siRNAs) to be used as gene-silencing triggers are chemical synthesis and vector expression of short hairpin RNAs. A more recent, alternative approach is the production of endoribonuclease-prepared short interfering RNAs (esiRNAs) by enzymatic digestion of long dsRNA with bacterial RNase III or recombinant Dicer. The present paper presents an algorithm for predicting the region for esiRNA synthesis that is optimal with regard to silencing efficacy and specificity. Using this algorithm, the optimal target regions for all protein-coding transcripts from the human, mouse, and rat genomes are presented. An open-access database for retrieval of this information has been established. A library of human esiRNAs has also been made available through the nonprofit German Genome Resource Center. Comparison of a sample of esiRNAs with chemically synthesized siRNAs indicates comparable efficacy in gene silencing, but a 12-fold reduction in the down-regulation of off-target transcripts as detected by microarray analysis.

CARBOHYDRATE AND GLYCOPROTEIN ANALYSIS

Chen S, LaRoche T, Hamelinck D, Bergsma D, Brenner D, Simeone D, Brand RE, Haab BB. Multiplexed analysis of glycan variation on native proteins captured by antibody microarrays. Nature Methods. 2007;4:437–444. [PubMed]

This method permits information about glycan structures to be ascertained for proteins captured on antibody microarrays, thereby allowing multiplexed analysis of large numbers of different proteins captured directly from biological samples. Lectin-binding profiles for the captured proteins are constructed using multiple lectins with diverse specificity as detection probes. To prevent the lectins from binding to the glycan moieties of the capture antibodies on the array, a chemical derivatization is employed. Cis-hydroxyl groups of the glycans on the antibodies are gently oxidized to aldehydes, then reacted with a bifunctional cross-linking reagent, and finally combined with a dipeptide to render the modified carbohydrate unrecognizable by the lectin probes. Using this system, alterations in the glycan alterations are described on the proteins MUC1 and CEA in the serum of patients with pancreatic cancer.

MACROMOLECULAR SYNTHESIS

Liu W, Brock A, Chen S, Chen S, Schultz PG. Genetic incorporation of unnatural amino acids into proteins in mammalian cells. Nature Methods. 2007;4:239–244. [PubMed]

Building upon this group’s success in accomplishing efficient, site-specific incorporation of diverse, unnatural amino acids into proteins in E. coli and yeast, this paper extends the methodology to proteins expressed in mammalian cells. Like the previous work, the method represents an extension of the genetic code relying upon a mutant aminoacyl-tRNA synthetase (aaRS) that can aminoacylate a cognate tRNA with the unnatural amino acid of choice. An E. coli aaRS previously evolved in yeast to perform this task with high fidelity is here transferred into mammalian cells. The tRNA is an amber suppressor tRNA from Bacillus, and incorporates the unnatural amino acid in response to an amber nonsense codon in the target gene. The system is shown to incorporate six different analogs of tyrosine into GFP reporter protein with high efficiency in CHO cells. This work is of interest in the context of research on in-vivo protein interactions, protein structure, and localization, as well as the production of potential gene therapy agents.

Durek T, Torbeev VY, Kent SBH. Convergent chemical synthesis and high-resolution x-ray structure of human lysozyme. Proceedings of the National Academy of Sciences, U.S.A. 2007;104:4846–4851.

Native chemical ligation has proved to be a powerful tool for the synthesis of proteins from chemically synthesized peptides. The procedure works by linking together unprotected peptide segments by reaction of an α-thioester on one its would-be neighbor, and the subsequent spontaneous rearrangement of the resulting thioester-linked intermediate to give a native peptide bond. The procedure has been used mostly for linking together only two peptide segments, and the length of proteins synthesized has therefore been restricted to about 100 amino acids. The present paper describes the use of a modified procedure that permits the synthesis of lysozyme (130 residues). In the modified procedure, called kinetically controlled ligation, a peptide thioarylester is reacted with a Cys-peptide thioalkylester in the absence of added thiol catalyst to yield a single product. Cysteines are chosen for the ligation chemistry from among the eight in the protein to allow four peptides of similar length (29–35 amino acids) to be employed. The two N-terminal peptides are linked together, then the two C-terminal peptides, and finally the complete molecule is assembled from the resulting halves. The synthesis is therefore fully convergent, i.e., each starting peptide is about the same number of chemical transformations away from the final product—a situation that provides better yields than sequential assembly tactics and more convenient production of variants with widely scattered substitutions. The resulting protein is catalytically active, and produces a high-resolution structure by x-ray crystallography.

METABOLOMICS

Cha S, Yeung ES. Colloidal graphite-assisted laser desorption/ionization mass spectrometry and MSn of small molecules. 1. Imaging of cerebrosides directly from rat brain tissue. Analytical Chemistry. 2007;79:2373–2385. [PubMed]

Cerebrosides are difficult to detect in crude extracts of eukaryotic tissues or in tissue imaging by MALDI MS because phosphatidylcholines (PCs) dominate the spectrum and produce signals that overlap those of cerebrosides. Study of ceramides therefore generally requires prior separation from PCs. Here, colloidal graphite in 2-propanol is used as a matrix for laser desorption/ionization and shown to provide strongly preferential ionization of ceramides without prior separation. In negative ion mode, the method supports detection of sulfatides in brain lipid extracts comparable to that of MALDI. Images of ceramides and sulfatides are acquired from rat brain tissue, including images of specific product ions from isobaric species.

MASS SPECTROMETRY

Fletcher JS, Lockyer NP, Vaidyanathan S, Vickerman JC. TOFSIMS 3D biomolecular imaging of Xenopus laevis oocytes using buckminsterfullerene (C60) primary ions. Analytical Chemistry. 2007;79:2199–2206. [PubMed]

This paper describes with an a proof-of-principal study dem- N-terminal onstrating for the first time the ability to collect high-resolution, three-dimensional images of biological samples by time-of-flight secondary ion mass spectrometry. The primary ion beam consists of buckminsterfullerene cations (C60+). These provide high yields of secondary ions for imaging, while causing minimal chemical damage to target molecular layers immediately below those yielding the desorbed ions. Information about depth distribution of molecules in the target is therefore amenable to acquisition as bombardment desorbs successive molecular layers of sample. Single freeze-dried Xenopus laevis oocytes are here chosen for imaging. In the positive ion mode, information about the depth distribution of cholesterol (m/z 369) and other lipids (m/z 540–570 and 800–1000) is recorded, and, in the negative ion mode, fatty acid side chains of lipids (m/z 255) are analyzed.

Edelson-Averbukh M, Pipkorn R, Lehmann WD. Analysis of protein phosphorylation in the regions of consecutive serine/threonine residues by negative ion electrospray collision-induced dissociation. Approach to pinpointing of phosphorylation sites. Analytical Chemistry. 2007;79:3476–3486. [PubMed]

Although MS in the negative ion mode is commonly used for detection of peptide phosphorylation, the positive ion mode is strongly favored for phosphopeptide sequencing because collision-induced dissociation (CID) produces negative ion MS/MS spectra that are difficult to interpret. This paper, however, demonstrates an especially useful feature of negative ion CID: it favors N–Cα backbone cleavage at phosphoserine(pSer)/phosphothreonine (pThr) residues to give abundant [zn − H3PO4] fragments. The extent of the dissociation is significantly higher than that of non-phosphorylated Ser/Thr residues. Moreover, sequences with consecutive Ser/Thr residues provide stronger z-type ions than isolated Ser or Thr. This enables assignment of the position of phosphorylation within Ser/Thr clusters, a task that is difficult to perform in positive ion CID because of suppression of backbone cleavages within such regions.

PROTEINS—PURIFICATION AND CHARACTERIZATION

Navratilova I, Papalia GA, Rich RL, Bedinger D, Brophy S, Condon B, Deng T, Emerick AW, Guan H-W, Hayden T, Heutmekers T, Hoorelbeke B, McCroskey MC, Murphy MM, Nakagawa T, Parmeggiani F, Qin X, Rebe S, Tomasevic N, Tsang T, Waddell MB, Zhang FF, Leavitt S, Myszka DG. Thermodynamic benchmark study using Biacore technology. Analytical Biochemistry. 2007;364:67–77. [PubMed]

Twenty-two individuals participate in a study to assess the capabilities of Biacore instruments and their operators to measure thermodynamic constants for a molecular interaction. An enzyme, carbonic anhydrase, is immobilized on the sensor surface, and allowed to bind four different sulfonamide inhibitors. Data are collected at temperatures from 6°C to 36°C, and equilibrium dissociation constants and van’t Hoff enthalpies are calculated. The values are in good accord with those measured by isothermal titration calorimetry, thereby validating the use of the Biacore methodology for this application. The study also highlights maintenance issues that produce data contaminated with significant instrument noise or drift, and specific steps are suggested for remediation.

Zhao C, O’Connor PB. Removal of polyethylene glycols from protein samples using titanium dioxide. Analytical Biochemistry. 2007;365:283–285. [PubMed]

Contamination of proteins with polyethylene glycols (PEGs) used for purification or stabilization is a major problem when measuring protein mass by electrospray MS because PEGs ionize strongly and may suppress or obscure signals from the protein. A method is described for PEG removal using titanium dioxide (TiO2) microcolumns. A solution of a protein containing 20 μM PEG 4000 is allowed to bind to a TiO2 micropipette from New Objective, Inc., PEG is washed away, and the protein is eluted for electrospray analysis. Phosphoproteins are applied to the column in a solution containing 20 mg/mL of 2,5-dihydroxybenzoic acid (DHB) in 80% acetonitrile and 0.1% trifluoroacetic acid (TFA). They are washed with the same solution and then with the solution lacking DHB, and finally are eluted with 75 mM ammonium hydroxide in water. Non-phosphorylated proteins are applied in a solution containing 20 mg/mL DHB in 10% acetonitrile and 0.1% TFA. They are washed with the same solution, and then with the solution lacking DHB, and finally eluted with 80% acetonitrile plus 0.1% TFA. Removal of PEG is efficient and rapid.

PROTEOMICS

Elias JE, Gygi SP. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nature Methods. 2007;4:207–214. [PubMed]

This paper represents a discussion of the use of decoy sequence databases to estimate the rate of false-positive peptide assignment in MS-based proteomics experiments. The strategy entails creating a decoy database from the database used for protein identification (the target database) such that the composition of the decoy preserves the general characteristics of the target database yet the number of sequences shared with it is minimized. This is most easily achieved by reversing the target protein sequences. The assumptions upon which this strategy is based are that the incidence of sequences shared between target and decoy databases is low, and that false-positive assignments are equally likely with the target and the decoy database. These assumptions are validated. Methods for constructing decoy databases are compared, including reversal of protein sequences, reversal of tryptic peptide sequences, and construction of sequences by stochastic methods. Each approach is found to yield a similar estimate for false-positive frequency.

Petyuk VA, Qian W-J, Chin MH, Wang H, Livesay EA, Monroe ME, Adkins JN, Jaitly N, Anderson DJ, Camp DG, II, Smith DJ, Smith RD. Spatial mapping of protein abundances in the mouse brain by voxellation integrated with high-throughput liquid chromatography–mass spectrometry. Genome Research. 2007;17:328–336. [PubMed]

The spatial distribution of proteins in mouse brain is investigated by dissecting a coronal brain section into 1-mm2 cubes (voxels) and then subjecting each to LC/MS analysis using a high-resolution Fourier transform ion cyclotron resonance mass spectrometer; 0.875 μg of protein from each cube is analyzed following trypsin digestion. An equal amount of a 18O-labeled peptide mixture from the entire coronal section of another mouse is added to each sample as an internal standard for quantitation. Seventy-one voxel samples are analyzed in all. In one week of data acquisition, the distribution of 1028 proteins is mapped. The penetration of the proteome by this method is superior to that of MALDI imaging approaches, although the spatial resolution is limited.

Dengjel J, Akimov V, Olsen JV, Bunkenborg J, Mann M, Blagoev B, Andersen JS. Quantitative proteomic assessment of very early cellular signaling events. Nature Biotechnology. 2007;25:566–568.

An automated, continuous quench flow system is described that allows cells to be trapped in states representing periods of as little as 1 sec post-stimulation. Three peristaltic pumps deliver cells, stimulus, and quenching reagent respectively. In the present case, cells in suspension are stimulated with epidermal growth factor (EGF). Quenching is accomplished with 70% ethanol at −20°C, a widely used fixing solution. The time following stimulation is controlled by varying the flow rate and the length of the tube between the mixing chamber and the point of delivery of the quenching reagent. After quenching, the phosphorylation status of selected proteins can be interrogated by immunoprecipitation and mass spectrometry. Cells that grow attached to a substratum can be analyzed in a similar way with the use of microcarrier beads.

Yi J, Kim C, Gelfand CA. Inhibition of intrinsic proteolytic activities moderates preanalytical variability and instability of human plasma. Journal of Proteome Research. 2007;6:1768–1781. [PubMed]

The changes in MALDI peptide profiles of serum and plasma that occur ex vivo through the action of endogenous proteases are documented. The stability of peptide profiles in serum and plasma are ascertained, and, in the case of plasma, the effects of anticoagulants (EDTA, sodium citrate, and heparin) and of protease inhibitors are described. Peptides that change during incubation are identified by MALDI-TOF/TOF and ESI-MS/MS analysis, and the changes are ascribed to the activation of the kallikrein-kinin system and the complement pathway, and to the subsequent action of aminopeptidases and carboxypeptidases. Proteolytic degradation occurs both in serum and plasma, but is more extensive in serum. A proprietary mixture of protease inhibitors that is used in the BD Diagnostics P100 EDTA-plasma-collection tube is shown to inhibit endogenous plasma proteases and peptidases, and to reduce the changes that occur in plasma peptides with time. Investigators are encouraged to add protease inhibitors to blood immediately upon blood collection to stabilize the plasma proteome for analysis.

MICROARRAYS

Zhang Z, Hesselberth JR, Fields S. Genome-wide identification of spliced introns using a tiling microarray. Genome Research. 2007;17:503–509. [PubMed]

Introns are removed from pre-mRNAs by the spliceosome prior to translation and are released in the form of lariats. Lariat DNA must be debranched before it can be degraded by exonucleases, and the debranching is performed in yeast by a debranching RNA endonuclease, Dbr1. In mutants lacking this enzyme, lariat RNA accumulates to high levels. In this study, a tiling array is used to compare the mutant and wild-type yeast strains in order to detect intronic RNA on a genome-wide scale. Yeast is a favorable test system for this study because introns occur in only 5% of the genes and possess a nearly invariant sequence at the branch-point. The approach identifies 141 of the 272 known introns. It confirms three previously predicted introns, and predicts four new ones. The introns that remain undetected are from genes expressed at low levels. The methodology is of interest for the detection of novel and alternatively spliced forms of eukaryotic genes generally.

FUNCTIONAL GENOMICS AND PROTEOMICS

Rinner O, Mueller LN, Hubálek M, Müller M, Gstaiger M, Aebersold R. An integrated mass spectrometric and computational framework for analysis of protein interaction networks. Nature Biotechnology. 2007;25:345–352.

This paper provides a framework for the analysis of protein interactions detected by co-immunoprecipitation (Co-IP). Complexes containing the human forkhead transcription factor, FoxO3A, are used as the test system. The general strategy for distinguishing specific from nonspecific interactions in Co-IP experiments is to compare quantitatively the proteins identified in Co-IPs from those in control and test cell lines. Proteins are identified by LC-MS/MS analysis using data-dependent product on acquisition schemes; in label-free experimental schemes, and quantitation is attempted by counting numbers of peptides assigned to the proteins identified. The stochastic nature of the data acquisition process makes for a high level of uncertainty in discriminating specifically from nonspecifically bound proteins in this scheme. In the present study, quantitation is performed instead on the basis of precursor ion signal strength. Signal strengths are measured in a series of mixtures between test and control Co-IPs in which the test is spiked into the control at varying levels. Nonspecific proteins are expected to remain at the same levels in this series of mixtures, whereas specific proteins produce signals whose intensity tracks with the amount spiked in. When a peptide is recognized to be of interest in this way, it may be targeted for MS/MS analysis to enable assignment. A similar scheme is used to identify changes in the composition of molecular complexes with alterations in the physiological state of the cell, as well as changes in the state of phosphorylation of the interacting proteins.

Stoevesandt O, Köhler K, Wolf S, André T, Hummel W, Brock R. A network analysis of changes in molecular interactions in cellular signaling. Molecular and Cellular Proteomics. 2007;6:503–513. [PubMed]

This report provides a highly parallel method for detecting signaling-dependent changes in patterns of molecular interactions. The concept is to construct peptide microarrays comprising known interaction motifs on signaling proteins. From lysates of resting and stimulated cells, complexes with which those signaling proteins interact are then captured on the array. This effectively represents a highly parallelized version of the co-immunoprecipitation strategy. The formation of a complex upon signaling is observed in this system either as a decrease in the binding of a protein due to the masking of its binding site for a peptide on the array, or as an increase in the binding of a protein owing to its interaction with another protein that binds to the array. The dissociation of a complex upon signaling leads to the opposite changes. The architecture of the bound complexes is amenable to analysis by adding peptides corresponding to interaction motifs on the constituent proteins and observing what combination of proteins dissociates as a result of the competitive challenge. The concept is tested by analysis of interactions involved in T-cell signal transduction.

MICROSCOPY

Verveer PJ, Swoger J, Pampaloni F, Greger K, Marcello M, Stelzer EH. High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy. Nature Methods. 2007;4:311–313. [PubMed]

Single-plane illumination microscopy (SPIM) is a new method of optical sectioning in which the sample is illuminated with a sheet of light, and only the plane that is in focus is illuminated, thus minimizing photo-damage and permitting the acquisition of many planes in the sample for three-dimensional imaging. In this paper, a deconvolution algorithm for use with SPIM is reported with which three-dimensional spatial resolution exceeding that of confocal fluorescence microscopy is achieved in large samples. The technique is used for rapid imaging of living specimens embedded in agar. Specimens can be large, including whole organisms and multicellular specimens produced by three-dimensional cell culture.

Dodt H-U, Leischner U, Schierloh A, Jährling N, Mauch CP, Deininger K, Deussing JM, Eder M, Zieglgänsberger W, Becker K. Ultramicroscopy: Three-dimensional visualization of neuronal networks in the whole mouse brain. Nature Methods. 2007;4:331–336. [PubMed]

A fixing method is described that permits light sheet illumination to be used for imaging opaque specimens such as mouse brain. The specimen is immersed in a medium with the same refractive index as protein, rendering the refractive index of intracellular structures the same as that of extracellular structures. This minimizes light scattering, and when no absorption also occurs, the specimen becomes transparent. Specimens as large as 2 cm can be cleared. Mouse embryos and the brains of mice up to postnatal day 10 are rendered completely transparent, allowing three-dimensional imaging with micrometer resolution.


Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities