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J Biomol Tech. 2007 April; 18(2): 135–140.
PMCID: PMC2062536

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.

AMINO ACID ANALYSIS AND SEQUENCING

Gogichaeva NV, Williams T, Alterman MA. MALDI TOF/TOF tandem mass spectrometry as a new tool for amino acid analysis. Journal of the American Society for Mass Spectrometry. 2007;18:279–284. [PubMed]

The fragmentation behavior of 34 different amino acids is investigated in a MALDI-TOF/TOF mass spectrometer under conditions of high-energy collision-induced dissociation, with a view to using characteristic fragments as indicator ions for amino acid quantitation. All the α-amino acids tested, except arginine and ornithine, give specific immonium ions as the major fragments, while the β-, γ-, and δ-amino acids do not produce immonium ions. Indicator ions allowing isomeric amino acids to be distinguished are observed. The data suggest that the methodology may be suitable for quantitative analysis of amino and organic acids.

DNA CHARACTERIZATION AND GENOTYPING

Ottesen EA, Hong JW, Quake SR, Leadbetter JR. Microfluidic digital PCR enables multigene analysis of individual environmental bacteria. Science. 2006;314:1464–1467. [PubMed]

The exploration of bacterial species diversity in naturally occurring microbial communities is rendered exceedingly difficult by the lack of ability to culture the vast majority of bacterial species in the laboratory. Although genes of interest can be isolated and sequenced, the bacterial species to which they belong have often resisted identification. This paper provides the means to overcome this problem. Microfluidic devices containing large numbers of small chambers (volume 6.25 nL) are used to isolate single cells or very small groups of cells by dilution. Several genes from the same cell can then be amplified and identified. The authors study the community of some 200 bacterial species living commensally in the hindgut of the California dampwood termite, enabling the insect to decompose wood. DNA was liberated from each well, and two genes were amplified: one a formyltetrahydrofolate synthase (FTHFS) that is species sequence specific, and the other a 16S rRNA gene that is generally distributed among bacteria, serving to confirm that a particular well actually contains a bacterial cell and to classify it. The FTHFS gene is shown by this method to be derived from a spirochete of the genus Treponema. The provision of techniques to link one or more genes to a particular bacterial species in a complex ecosystem is a landmark event in environmental research.

Von Mering C, Hugenholz P, Raes J, Tringe SG, Doerks T, Jensen LJ, Ward N, Bork P. Quantitative phylogenetic assessment of microbial communities in diverse environments. Science. 2007;315:1126–1130. [PubMed]

A traditional approach to the taxonomic profiling of microbial communities relies on polymerase chain reaction (PCR) to amplify ribosomal RNA. This requires specific primers or sequence anchors, and therefore presupposes some knowledge of the bacteria that are present. The use of PCR further carries a risk of quantitative distortion. These limitations are circumvented in the methodology described in the present paper, in which shotgun sequencing of community DNA (so-called metagenomics) provides a more direct and less biased view of uncultured cells. Thirty-one genes known to be useful for phylogenetic analysis are mapped from four diverse environments. The results show that some communities evolve faster than others, and that certain bacterial clades show long-lasting habitat preferences.

Litos IK, Ioannou PC, Christopoulos TK, Traeger-Synodinos J, Kanavakis E. Genotyping of a single-nucleotide polymorphism by primer extension reaction in a dry-reagent dipstick format. Analytical Chemistry. 2007;79:395–402. [PubMed]

A low-cost SNP assay that can be performed without specialized instrumentation or technical training is developed for use in clinical diagnostic laboratories. It is based on primer extension and the products are read using a dry-reagent dipstick. Genomic DNA spanning the region of interest is amplified by PCR. Two primer extension reactions are performed with allele-specific primers corresponding to the alleles to be discriminated. These contain oligo(dA) at the 5′ end. Biotin dUTP is incorporated into the extended strand. The product is applied to the strip, and the strip is immersed in buffer. The DNA moves along the strip by capillary action, and hybridizes to oligo(dT)-gold nanoparticles. The extended products are captured by steptavidin immobilized in the test zone on the strip, and a red line appears. A second red line is produced in a control zone on the strip by hybridization of the nanoparticles with immobilized oligo(dA). This assay is validated by genotyping the genomic DNA from 217 patients for six SNPs in the mannose-binding lectin gene.

CARBOHYDRATE AND GLYCOPROTEIN ANALYSIS

Bernhard OK, Kapp EA, Simpson RJ. Enhanced analysis of the mouse plasma proteome using cysteine-containing tryptic glycopeptides. Journal of Proteome Research. 2007;6:987–995. [PubMed]

To improve existing procedures for affinity capture of glycopeptides, this paper incorporates a second affinity separation step to enhance capture of low-abundance species. Plasma proteins are coupled via their carbohydrate moieties to a resin using hydrazide chemistry. Cysteine residues are then biotinylated. After digesting with trypsin and washing to remove non-glycosylated peptides, tethered N-glycosylated peptides are released by digestion with protein N-glycanase F. Biotinylated peptides are then selected by affinity capture with monomeric avidin, and identified by LC/MS. Using this procedure, 51 proteins (104 glycosylation sites) are identified from samples of 200 μL of plasma, including low-abundance species such as growth factors, not detected without the biotin selection step.

Sun B, Ranish JA, Utleg AG, White JT, Yan X, Lin B, Hood L. Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics. Molecular and Cellular Proteomics. 2007;6:141–149. [PubMed]

This work also exploits selective capture on a solid support using hydrazide chemistry and release with protein N-glycanase F. However, the glycoproteins are digested with trypsin prior to carbohydrate capture to improve the solubility of membrane proteins and expose all sites of carbohydrate capture to access to the capture reagents. This procedure is used to study the microsomal fraction of an ovarian cancer cell line, and results in the identification of 136 proteins in a single LC/MS experiment.

METABOLOMICS

Böttcher C, von Roepenack-Lahaye E, Willscher E, Scheel D, Clemens S. Evaluation of matrix effects in metabolite profiling based on capillary liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. Analytical Chemistry. 2007;79:1507–1513. [PubMed]

This study addresses the concern that matrix effects may limit the reliability of metabolite quantification in untargeted metabolomic studies performed by electrospray ionization mass spectrometry. Matrix effects are measured by post-extraction addition of reference compounds to different complex biological matrices, by post-column infusion of reference compounds, and by mixing complex matrices together. While significant matrix effects are observed when comparing very divergent samples, these effects contribute little to measurements of changes in metabolite levels unless vastly different matrices are compared. The methodology described in this paper will be useful for assessing matrix effects in studies involving new extraction schemes or samples of new biological origin as part of the essential validation of metabolite profiling studies.

PEPTIDE AND PROTEIN SYNTHESIS

Vogel EM, Imperiali B. Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies. Protein Science. 2007;16:550–556. [PubMed]

“Caged” phosphoamino acids have side chains that release a phosphorylated species upon photolysis. The ability to create a phosphorylated species on demand in this way enables the function of specific phosphate residues to be ascertained in time-controlled experiments. This paper reports the incorporation of a “caged” phosphotyrosine residue into the intact 61-kDa protein paxillin, which is involved in the regulation of signaling cascades at sites of focal adhesion between cells and the extracellular matrix. Paxillin variants are constructed in a semisynthesis scheme involving native chemical ligation of synthetic peptides corresponding to residues 2–36 of the intact protein, and residues 37 557 expressed by recombinant methods. Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-tyrosine is incorporated as the caged phosphotyrosine in the chemically synthesized section to produce an intact protein with the unnatural residue at position 31. The incorporation of caged residues into complex targets that can then be introduced into cells by microinjection is expected to facilitate functional analysis of specifically phosphorylated protein species.

Okuyama M, Laman H, Kingsbury SR, Visintin C, Leo E, Eward KL, Stroeber K, Boshoff C, Williams GH, Selwood DL. Small-molecule mimics of an α-helix for efficient transport of proteins into cells. Nature Methods. 2007;4:153–159. [PubMed]

Some intact proteins can be internalized by live cells through their possession of peptide sequences with transduction properties. These sequences are amphipathic helices containing several arginine residues. Peptides with these properties have been used to mediate the internalization of various cargoes, but their usefulness is limited by their metabolic instability. The present paper describes a non-peptidic, small-molecule analog of an alpha-helical protein transduction domain, and demonstrates its use in delivering the anti-proliferative protein geminin into human cancer cells in functionally active form.

MASS SPECTROMETRY

Swaney DL, McAllister GC, Wirtala M, Schwartz JC, Syka JEP, Coon JJ. Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors. Analytical Chemistry. 2007;79:477–485. [PubMed]

Electron transfer dissociation (ETD) has crucial advantages over collision-induced dissociation (CID) for generating product ions informative for peptide sequence on a chromatographic timescale. However, the efficiency with which product ions are produced in ETD is low for doubly protonated peptide ions, the most commonly encountered ions generated by electrospray ionization of tryptic peptides. The ETD process for these precursors instead favors charge reduction without cleavage. In this paper, the charge-reduced products of ETD are subjected to low-energy CID, and are shown to fragment with high efficiency. Moreover, the products are the same c- and z-type fragments typical of ETD. The hybrid ETD/CID technique can be performed without special modifications to the instrumentation, and implementation adds a relatively short time to the duration of each scan.

Sun S, Meyer-Arendt K, Eichelberger B, Brown R, Yen C-Y, Old WM, Pierce K, Cios KJ, Ahn NG, Resing KA. Improved validation of peptide MS/MS assignments using spectral intensity prediction. Molecular and Cellular Proteomics. 2007;6:1–17. [PubMed]

Methodology for validating peptide assignments made by automated search routines is described, based on two criteria commonly used when inspecting data manually. The first criterion is consistency of fragment ion intensities with predicted gas-phase chemistry. Consistency is assessed by comparing spectral data with theoretical low-energy CID spectra calculated with the MassAnalyzer software of Zhang Z (Anal Chem 76;2004:3908–3922). The second criterion is whether a high proportion of the total ion current in MS/MS spectra can be derived from the matched peptide. This methodology supports better discrimination between correct and incorrect assignments than SEQUEST XCorr scoring or MASCOT Mowse scoring. Triply charged peptides show especially strong improvement, and singly charged ions show moderate improvement. The new scoring routines also reveal chimeric spectra derived from simultaneous fragmentation of two or more different peptides.

PROTEINS—PURIFICATION AND CHARACTERIZATION

Zilberstein G, Korol L, Antonioli P, Righetti PG, Bukshpan S. SDS-PAGE under focusing conditions: An electrokinetic transport phenomenon based on charge neutralization. Analytical Chemistry. 2007;79:821–827. [PubMed]

A new form of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is described, in which the gel is made with a gradient of increasing positive charge density from the cathodal to the anodal end. The gradient is made by pouring a gel of uniform porosity but using a two-vessel gradient mixer to produce a gradient of basic Immobiline. Negatively charged SDS-protein complexes migrate through such a gel until they encounter a density of positive charge sufficient to stop migration. In this way, larger proteins, which bind larger numbers of SDS monomers and therefore bear higher negative charge, migrate further than smaller proteins. This is the inverse of their behavior in conventional SDS-PAGE. The immobilized charge gradient is made by design to produce a linear, rather than the conventional logarithmic, plot of Mr vs. migration distance. This system has several distinct advantages. It is capable of separating peptides and small proteins that are below the size range typically resolved by conventional SDS-PAGE. Also, by creating plateaus in the charge gradient, the separation of species with very similar Mr values can be enhanced. Resolution of molecules differing by only 150 Da is demonstrated. It is anticipated that the method will also be useful for separating DNA molecules.

Pitre A, Pan Y, Pruett S, Skalli O. On the use of ratio standard curves to accurately quantitate relative changes in protein levels by Western blot. Analytical Biochemistry. 2007;361:305–307. [PubMed]

Western blotting is frequently used to measure changes in expression level of selected proteins. The ratios of densitometric measurements are usually assumed to provide accurate values for the ratios of amounts of the proteins present. This paper, however, shows that this is not generally true, because the straight line of best fit for data displayed in plots of densitometry value vs. total cellular protein often do not extrapolate to zero densitometry signal for zero protein loaded. This problem can be circumvented by plotting a standard densitometry curve using a purified protein standard. If such a standard is unavailable, a ratio standard curve can be used in which densitometric ratios are plotted against known ratios of total cellular protein.

Savchenko A, Kashuba E, Kashuba V, Snopok B. Imaging technique for the screening of protein-protein interactions using scattered light under surface plasmon resonance conditions. Analytical Chemistry. 2007;79:1349–1355. [PubMed]

Although single-channel surface-plasmon resonance (SPR) systems have found extensive use for measuring protein-protein and protein–small molecule interactions, they have low throughput capacity and require large amounts of material for analysis. The work described in the present paper is aimed at constructing a microarray SPR spectrometer capable of detecting hundreds of concurrent interactions. This is accomplished by measuring scattered rather than reflected light under SPR conditions. The maximum scattering angle is shown to correlate with the traditionally employed reflection minimum. A panoramic scanning spectrometer is used to record both variations in volume refractive index and protein layer formation on a metal surface. The method is validated by detecting interactions between protein A and commercial antibodies or human serum in microarray format. The instrument design is simple and low cost. The method increases dynamic range and integrates data with those from surface plasmon field-enhanced fluorescence spectroscopy.

Sprangers R, Kay LE. Quantitative dynamics and binding studies of the 20s proteasome by NMR. Nature. 2007;445:618–622. [PubMed]

Conformational studies of proteins by proton nuclear magnetic resonance (NMR) have traditionally been restricted to small proteins, because resolution diminishes with increasing protein size as the number of proton signals increases, and because the NMR signals are weaker due to more rapid transverse decay. The present milestone study demonstrates the extension of proton NMR to the conformational analysis of a protein of 670 kDa, the 20S archaebacterial proteasome. The methodology addresses the problem of resolution by restricting the collection of signals to the protons in just one of the methyl groups in valines and the isopropyl unit of leucines. The protons in the other methyl unit in leucines or valines are rendered invisible by replacing them with deuterons, as are the protons in all other amino acids in the protein. The problem of signal strength is addressed by using transverse relaxation-optimized spectroscopy methods. Using these techniques, molecular motions important in permitting access of polypeptide substrates to the enzyme’s catalytic centers are documented.

PROTEOMICS

Gundry RL, Jelinek CA, Van Eyk JE, Cotter RJ. Investigation of an albumin-enriched fraction of human serum and its albuminome. Proteomics Clinical Applications. 2007;1:73–88.

Because albumin removal is a process routinely incorporated into proteomic studies of serum and plasma, there is widespread concern that other proteins are removed along with albumin, either because they are bound to or co-purify with albumin. This study is a systematic survey to identify such proteins. Antibody affinity chromatography and size exclusion chromatography are used as alternative methods for albumin removal. Proteins removed with albumin are fractionated by reverse-phase chromatography and SDS-PAGE and then identified by mass spectrometry. The intact molecular weight of the proteins is also measured to assess whether they represent whole proteins or proteolytic fragments. Thirty-five proteins are identified, of which 24 are intact. They include both high- and low-abundance proteins.

Aristoteli LP, Molloy MP, Baker MS. Evaluation of endogenous plasma peptide extraction methods for mass spectrometric biomarker discovery. Journal of Proteome Research. 2007;6:571–581. [PubMed]

Three different methods are compared for their efficacy in extracting peptides from plasma for peptidomic studies: solid-phase extraction, acetonitrile precipitation, and size exclusion. Solid-phase extraction using Waters Oasis HLB media provides the largest number of peptides detectable by MALDI mass spectrometry and the greatest diversity of molecular weight, even though the method also yields a higher level of residual protein. As expected, the majority of the peptides recovered represent proteolytic degradation products of plasma proteins. Interestingly, different extraction methods yield unique mass signals, suggesting that the best peptidome coverage may require use of a combination of extraction techniques.

Chi A, Huttenhower C, Geer LY, Coon JJ, Syka JEP, Bai DL, Shabanowitz J, Burke DJ, Troyanskaya OG, Hunt DF. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proceedings of the National Academy of Sciences, U.S.A. 2007;104:2193–2198.

Molina H, Horn DM, Tang N, Mathivanan S, Pandey A. Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proceedings of the National Academy of Sciences, USA. 2007;104:2199–2204.

These two papers describe global profiling of phosphorylation sites using electron transfer dissociation (ETD) mass spectrometry. Chi et al. begin with total yeast cell proteins, digest with endoproteinase Lys-C, and use immobilized metal ion affinity chromatography for phosphopeptide purification. They identify 1252 phosphorylation sites on 629 proteins, and note that kinases themselves account for a disproportionate number of these. Molina et al. begin with human 293T cells, and purify phosphopeptides using titanium oxide. They identify 1435 phosphorylation sites on 500 proteins; 80% of these 1435 had not been described previously. Molina et al. note that ETD produced 60% more phosphopeptide identifications than collision-induced dissociation, with an average of 40% more fragment ions, which facilitate identification and phosphate localization.

Mallick P, Schirle M, Chen SS, Flory MR, Lee H, Martin D, Ranish J, Raught B, Schmitt R, Werner T, Kuster B, Aebersold R. Computational prediction of proteotypic peptides for quantitative proteomics. Nature Biotechnology. 2007;25:125–131.

The complexity of peptide mixtures created by digesting entire proteomes is such as to overwhelm the ability of even our best analytical procedures to perform in-depth proteomic analysis in a reasonable timeframe. The level of complexity can be greatly reduced if analysis is restricted to just one or a few peptides that uniquely identify each protein, rather than allowing instruments to identify large numbers of peptides from just a few proteins over and over again. This paper addresses the problem of assembling a list of such diagnostic, or “proteotypic,” peptides, and then, by identifying the characteristics they share, predicting which peptides from previously unobserved proteins are likely to be proteotypic. From large, well-curated archives or yeast proteomic data acquired using the most commonly employed proteomic platforms, the peptides most commonly observed from each identified protein are listed as proteotypic peptides. Their physico-chemical properties are then evaluated, and lists of the characteristics most predictive of proteotypic peptides are computed. These sets of characteristics are then shown to predict proteotypic peptides from the proteins of any organism on like proteomic platforms with greater than 85% cumulative accuracy. The availability of proteotypic peptide lists is expected to have a wide-ranging impact, including rational selection of peptide targets to quantify selected proteins, validation of protein identifications, and characterization of the physical principles governing key features of proteomic workflows that influence which peptides are observed.

Lam H, Deutsch EW, Eddes JS, Eng JK, King N, Stein SE, Aebersold R. Development and validation of a spectral library searching method for peptide identification from MS/MS. Proteomics. 2007;7:655–667. [PubMed]

A library of 30,000 MS/MS spectra for peptides from proteins of Saccharomyces cerevisiae is announced that can be used for rapid and efficient peptide assignment by spectral library searching. For spectral matching, an open-source search tool, SpectraST, is described. This tool vastly outperforms SEQUEST in speed and ability to discriminate correct and incorrect matches. The library searching method is particularly well suited for targeted proteomics applications.

MICROARRAYS

Fiegler H, Geigl JB, Langer S, Rigler D, Porter K, Unger K, Carter NP, Speicher MR. High resolution array-CGH analysis of single cells. Nucleic Acids Research. 2007;35:e15. [PubMed]

In order to assess the heterogeneity in genome copy number in tumors, DNA is prepared from individual cultured cells or cells from tumors for comparative genomic hybridization (array-CGH). DNA amplification is performed with the GenomePlex Single Cell Whole Genome Amplification Kit from Sigma-Aldrich. Single copy changes as small as 8.3 Mb are detected in single cells by this method.

Yu H, Nguyen K, Royce T, Qian J, Nelson K, Snyder M, Gerstein M. Positional artifacts in microarrays: Experimental verification and construction of COP, an automated detection tool. Nucleic Acids Research. 2007;35:e8. [PubMed]

In experiments with spotted microarrays, correlations in the expression of different genes may arise as artifacts of chip design through carryover of DNA during the arraying process. This paper provides evidence for two such artifacts. In the first, genes that are close to one another in an array sometimes show higher correlation in their expression profiles than genes further away. The results of experiments to test the extent of carryover of DNA on arrayer pins during the printing process suggest that such carryover may account for these correlations. In the second artifact, correlations exist between genes that were close together on the microtiter plate in which the DNA was stored prior to printing. This is presumed to be due to cross-contamination during sample preparation and the polymerase chain reaction process. Its magnitude is smaller than that of the first artifact, but is sometimes non-negligible. A software tool, COP (COrrelations by Positional artifacts) is introduced to detect these artifacts in microarray experiments. It is made available integrated with the microarray data normalization tool, ExpressYourself, at http://bioinfo.mbb.yale.edu/ExpressYourself/.

FUNCTIONAL GENOMICS AND PROTEOMICS

Spielman RS, Bastone LA, Burdick JT, Morley M, Ewens WJ, Cheung VG. Common genetic variants account for differences in gene expression among ethnic groups. Nature Genetics. 2007;39:226–231. [PubMed]

The analysis of the polymorphisms that produce variation in gene expression is here extended to consideration of differences in the incidence of such polymorphisms in individuals from different human populations. Variation in gene expression is characterized in cells from individuals belonging to European-derived and two Asian-derived populations. In 1097 of 4197 genes tested, the incidence of quantitative phenotypes differs significantly between populations. In the case of phenotypes for which strong evidence exists for cis determinants of gene expression, most of the variation is due to allele frequency differences at cis-linked regulators. The results indicate that specific genetic variation between populations contributes appreciably to differences in gene expression phenotypes. Many complex diseases (e.g., diabetes) are believed to be influenced by levels of gene expression. Differences in the incidence of such diseases between populations may be due in part to allele frequency differences at regulatory loci of the kind highlighted in this paper.

Thomas RK, Baker AC, Debiasi RM, Winckler W, Laframboise T, Lin WM, Wang M, Feng W, Zander T, Macconnaill LE, Lee JC, Nicoletti R, Hatton C, Goyette M, Girard L, Majmudar K, Ziaugra L, Wong KK, Gabriel S, Beroukhim R, Peyton M, Barretina J, Dutt A, Emery C, Greulich H, Shah K, Sasaki H, Gazdar A, Minna J, Armstrong SA, Mellinghoff IK, Hodi Fs, Dranoff G, Mischel PS, Cloughesy TF, Nelson SF, Liau LM, Mertz K, Rubin MA, Moch H, Loda M, Catalona W, Fletcher J, Signoretti S, Kaye F, Anderson KC, Demetri GD, Dummer R, Wagner S, Herlyn M, Sellers WR, Meyerson M, Garraway LA. High-throughput oncogene mutation profiling in human cancer. Nature Genetics. 2007;39:347–351. [PubMed]

This paper represents a first step in systematically identifying the genetic changes responsible for tumor initiation and progression. High–throughput genotyping is performed to screen for 238 known mutations in 17 oncogenes across 1000 human tumor samples. Surprisingly, in 3 of the genes, no mutations are found, and in the remaining 14, only 30% bear mutations. The results indicate that recurrent mutations are less common than previously thought.

Hölzel M, Rohrmoser M, Orban M, Hömig C, Harasim T, Malamoussi A, Gruber-Eber A, Heissmeyer V, Bornkamm G, Eick D. Rapid conditional knock-down-knock-in system for mammalian cells. Nucleic Acids Research. 2007;35:e17. [PubMed]

This system facilitates the interpretation of RNAi experiments by replacing the knocked down protein by an ectopic protein that serves to preserve associated complexes and eliminate off-target effects. By replacing with mutant proteins, the roles of specific domains or modifications can also be ascertained. Two episomal vectors are used to achieve these results. A reconstitution vector allows doxycycline-dependent expression of the gene of interest (either wild type or mutant). A conditional knockdown vector expresses an siRNA embedded in an miRNA environment that targets the untranslated region of the endogenous RNA. This vector encodes a different resistance gene for selecting knock-down cells.

Schlüter H, Rykl J, Thiemann J, Kurzawski S, Gobom J, Tepel M, Zidek W, Linscheid M. Mass spectrometry–assisted protease substrate screening. Analytical Chemistry. 2007;79:1251–1255. [PubMed]

A screening method for identifying the natural substrates of proteases is described. Fractions of endogenous proteins are covalently immobilized on cyanogen bromide–activated Sepharose beads. The beads are washed to remove unbound material, and the protease of interest is then added in solution. After incubation, the solution, now containing proteolytically released peptides, is concentrated and desalted using reverse-phase tips and analyzed by MALDI mass spectrometry to identify the proteolytic products. The method is demonstrated by incubating immobilized human plasma proteins with thrombin and identifying the fibrinopeptides released from fibrinogen. The method can be readily automated, and is suitable also for protease inhibitor screening.


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