Selective Labeling Method Research Articles

Super-Resolution Fluorescence Imaging for Semiconductor Nanoscale Metrology and Inspection.

The increase in the number and complexity of process levels in semiconductor production has driven the need for the development of new measurement methods that can evaluate semiconductor devices at the critical dimensions of fine patterns and simultaneously inspect nanoscale contaminants or defects. However, conventional optical inspection methods often fail to resolve device patterns or defects at the level of tens of nanometers required for device development owing to their diffraction-limited resolutions. In this study, we used the stochastic optical reconstruction microscopy (STORM) technique to image semiconductor nanostructures with feature sizes as small as 30 nm and detect individual 20 nm-diameter contaminants. STORM imaging of semiconductor nanopatterns is based on the development of a selective labeling method of fluorophores for a negative silicon oxide surface using the charge interaction of positive polyethylenimine molecules. This study demonstrates the potential of STORM for nanoscale metrology and in-line defect inspection of semiconductor integrated circuits.

Reinvestigation of an O-Salicylaldehyde Ester Functional Group in Aqueous Buffer and Discovery of a Coumarin Scaffold Probe for Selective N-Terminal Cysteine Labeling.

Many intracellular proteins are metabolically unstable, and their half-life was known to be controlled by the "N-end rule," that is, the N-terminal residue controlled protein stability. To visualize or measure the cellular stability of a protein, depending on the N-terminal residues, attention is being paid to the development of selective labeling methods for individual N-terminal amino acids. However, there are only a limited number of functional groups available for specific N-terminal amino acid labeling in a biological environment. Herein, we report a re-examination of salicylaldehyde ester for selective N-terminal residue tagging. Salicylaldehyde ester has been used for chemical ligation to N-terminal serine or threonine under pyridine/acetic acid conditions. Inspired by previous selective serine/threonine labeling, N-terminal labeling of salicylaldehyde ester in aqueous buffer has been examined by using boron-dipyrromethene (BODIPY), rhodamine, and coumarin probes. Surprisingly, the selectivity not only significantly differed, depending on the fluorophore incorporated in salicylaldehyde, but was also perturbed by the addition of a small fraction of phosphate-buffered saline. In particular, the coumarin-based salicylaldehyde ester probe showed notable selectivity against N-terminal cysteine under aqueous buffer conditions. This result reveals the serendipitous discovery of a new N-terminal cysteine labeling strategy.

Detection of mismatched 5-hydroxymethyluracil in DNA by selective chemical labeling

How DNA demethylation is achieved in mammals is still under extensive investigation. One proposed mechanism is deamination of 5-hydroxymethylcytosine to form 5-hydroxymethyluracil (5hmU), followed by base excision repair to replace the mismatched 5hmU with cytosine. In this process, 5hmU:G mispair serves as a key intermediate and its localization and distribution in mammalian genome could be important information to investigate the proposed pathway. Here we describe a selective labeling method to map mismatched 5hmU. After converting other cytosine modifications to 5-carboxylcytosines, a biotin tag is installed onto mismatched 5hmU through β-glucosyltransferase-catalyzed glucosylation and click chemistry. The enriched 5hmU-containing DNA fragments can be subject to subsequent sequencing to reveal the distribution of 5hmU:G mispair with base-resolution information acquired.

Efficient molecular and genetic technique for identification of Salmonella and Proteus strains

Fast microorganism genotyping technique has been developed with the use of the double distribution and selective labeling method (DDSL) for identification of disease-associated serotypes of Salmonella and Proteus strains. Isolates have been obtained from poultry tissues and dung from different places in various years. The results of strain genotyping conform to epizootological data. The method can be recommended for practical application in ascertainment of transmission of infectious disease and localization of bacterial pathogen source.

Chemical labeling of electrochemically cleaved peptides

Cleavage of peptide bonds C-terminal to tyrosine and tryptophan after electrochemical oxidation may become a complementary approach to chemical and enzymatic cleavage. A chemical labeling approach specifically targeting reactive cleavage products is presented here and constitutes a promising first step towards the development of a new proteomics workflow. Hexylamine was used to react with the spirolactone moieties generated after electrochemical oxidation and cleavage of tripeptides. The influence of pH and reaction time on the yield was determined and the excess of tagging reagent was optimized. Selective detection of the tagged cleavage products was achieved by precursor ion scanning in a triple quadrupole mass spectrometer. Optimal labeling was reached under aqueous conditions when working at pH 10 with a reaction time of 0.5 min. The excess of hexylamine over spirolactone groups can be significantly decreased by working under non-aqueous conditions in pure acetonitrile to prevent spirolactone hydrolysis. The specific formation of hexylamine-containing y(1) reporter ions generated by collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) allows for selective detection by precursor ion scanning of the cleaved and labeled peptides. This work presents a method for selective labeling and detection of electrochemically cleaved Tyr- and Trp-containing peptides for which reaction conditions have been optimized with hexylamine as labeling agent. This workflow offers new possibilities for electrochemical oxidation, cleavage and labeling of peptides and proteins.

A novel leaflet-selective fluorescence labeling technique reveals differences between inner and outer leaflets at high bilayer curvature

Understanding the differences in the physical properties of the inner and outer leaflet of membranes and how the leaflets are coupled to each other requires methods that can selectively label both the outer and inner leaflets. In this report we introduce a combined chromatography/cyclodextrin method for selective labeling of the inner leaflet. Combining this method with selective labeling of the outer leaflet, we are able to show that there is a distinct difference in polar headgroup physical properties of the inner and outer leaflet headgroups in small unilamellar vesicles composed of a wide variety of phosphatidylcholines and a phosphaticylcholine/sphingomyelin mixture. It appears that the inner leaflet headgroups are more tightly packed than those of the outer leaflet. This differential packing disappears when vesicle size increases, showing that it is a consequence of membrane curvature. Differential packing is also reduced as acyl chain length is decreased. In the future, selective leaflet labeling is likely to be a powerful tool for investigating the properties of asymmetric lipid vesicles.

Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells.

Most clinically approved biomarkers of cancer are glycoproteins, and those residing on the cell surface are of particular interest in biotherapeutics. We report a method for selective labeling, affinity enrichment, and identification of cell-surface glycoproteins. PC-3 cells and primary human prostate cancer tissue were treated with peracetylated N-azidoacetylgalactosamine, resulting in metabolic labeling of cell surface glycans with the azidosugar. We used mass spectrometry to identify over 70 cell surface glycoproteins and biochemically validated CD146 and integrin beta-4, both of which are known to promote metastatic behavior. These results establish cell-surface glycoproteomics as an effective technique for discovery of cancer biomarkers.

General method for selective labelling of double‐chain cysteine‐rich peptides with a lanthanide chelate via solid‐phase synthesis

The use of lanthanides in preference to radioisotopes as probes for various biological assays has gained enormous popularity. The introduction of lanthanide chelates to peptides/proteins can be carried out either in solution using a commercially available labelling kit or by solid-phase peptide synthesis using an appropriate lanthanide chelate. Herein, a detailed protocol for the latter is provided for the labelling of peptides or small proteins with diethylenetriamine-N, N, N″, N″-tetra-tert-butyl acetate-N'-acetic acid (DTPA) chelate or other similar chelates on a solid support using a chimeric insulin-like peptide composed of human insulin-like peptide 5 (INSL5) A-chain and relaxin-3 B-chain as a model peptide. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

Presence of α7 nicotinic acetylcholine receptors on dorsal root ganglion neurons proved using knockout mice and selective α‐neurotoxins in histochemistry

In complex tissues where multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed, immunohistochemistry has been the most popular tool for investigation of nAChR subunit distribution. However, recent studies with nAChR subunit knockout mice demonstrated that a large panel of antibodies is unsuitable. Thus, we aimed to develop a histochemical method for selective labeling of alpha7 nAChR with neurotoxins, utilizing alpha7 nAChR-transfected cells, dorsal root ganglia (DRG) and spinal cord from wild-type and knockout mouse. The specificity of Alexa Fluor 488-conjugated alpha-bungarotoxin (Alexa-alphaBgt) was demonstrated in binding to alpha7-transfected cells inhibited by long-chain alpha-cobratoxin (CTX), but not short-chain alpha-neurotoxin II (NTII). In contrast, binding to Torpedo muscle-type nAChRs and to motor end plates in mouse tongue sections was prevented by both CTX and NTII. In tissue sections of DRG, expressing all neuronal nAChR subunits, only CTX precluded Alexa-alphaBgt labeling of neurons, with no staining for alpha7 nAChR knockout tissue. It proved that alpha7 nAChRs are the major alphaBgt-binding sites in mouse DRG. Corresponding results were obtained for terminals in the spinal cord. Thus, we present a protocol utilizing Alexa-alphaBgt and non-labeled CTX/NTII that allows specific histochemical detection of alpha7 nAChR with a spatial resolution at the level of single axon terminals.

Chain Localization and Interfacial Thickness in Microphase-Separated Structures of Block Copolymers with Variable Composition Distributions

The effect of composition distribution on microphase-separated structures of AB diblock and BAB triblock copolymers was investigated at the molecular level by measurements of neutron reflectivity. Monodispersed three poly(styrene)-d8-block-poly(2-vinylpyridine) (DP) and three labeled triblock copolymers (PDP) and unlabeled counterparts were synthesized by a living anionic polymerization process designed to yield constant molecular weight with different volume fractions. The selective labeling method employs the blending of copolymers for a segmental distribution study while the labeled polymers are mixed for an interfacial study. Scattering length density profiles were observed by neutron reflectivity measurements for blended thin films produced by spin-coating and successive thermal annealing. It was confirmed that the blended thin films exhibit simple alternating lamellar structures, an observation consistent with the observed structures of bulk specimens. The localization phenomenon of longer and short...

A Combinatorial Selective Labeling Method for the Assignment of Backbone Amide NMR Resonances

A combinatorial selective labeling (CSL) method is presented for the assignment of backbone amide NMR resonances, which has a particular application in the identification of protein-ligand interaction sites. The method builds on the dual amino acid selective labeling technique. In the CSL method a number of different samples are produced, each with a different pattern of labeled amino acids. By analyzing peak intensities in HSQC and 2D HNCO spectra of these samples, a large number of combinations of amino acid pairs can be simultaneously assigned. We demonstrate the method on the 27 kDa protein GFP. The samples can be produced rapidly and cost-effectively in a commercially available in vitro translation system. The method greatly simplifies the process of backbone assignment and would be very straightforward to automate.

A novel 15N-labeling method to selectively observe 15NH 2 resonances of proteins in 1H-detected heteronuclear correlation spectroscopy

An experimental method to selectively label side-chain NH 2 groups of glutamine and asparagine in proteins with 15N is proposed. This selective labeling method enables to observe only 15NH 2 resonances and thus, to discriminate between 15NH and 15NH 2 resonances in a 1H-detected heteronuclear correlation spectrum. This method gives results with approximately two times higher sensitivity than those obtained by elaborate pulse sequences such as DEPT-HSQC and will be useful for studying the molecular interaction involving the side chains of Asn and Gln residues.

Selective labelling of lymphoid tissues by extracorporeal perfusion

A method for selective labelling of lymphoid tissues by means of an extracorporeal perfusion system is described. The applicability of this technique was tested by local labelling of a part of the small intestine with its mesenteric lymph nodes, spleen and bone marrow in normal young pigs. Tritiated thymidine was added to the perfusate and the selectivity of the labelling procedure tested by scintillation counting of serum samples, of specimens of lymphoid tissues and by autoradiography. There was only negligible spillover of the isotope and the perfused organs were well preserved.

The importance of the chemical state of 99 mTc radiopharmaceuticals: an effective tumor imaging form of 99 mTc bleomycin

99 m Tc-labeled bleomycin ( 99 m Tc-BLM) has been introduced as a tumor-imaging agent. Nevertheless, this complex has been misjudged and its value for clinical use has received no proper evaluation. Various 99 m Tc-BLM species labeled with 99 m Tc in different chemical states are produced depending upon the labeling conditions such as the concentrations of BLM and SnCl 2, pH and reaction time. As a consequence, their biological behavior is found to be markedly affected by the state of 99 m Tc in the labeled compound administered. Detailed studies of the labeling reactions lead us to conclude that of the complexes tested, a 99 m Tc-BLM complex in which tetravalent 99 m Tc is coordinated through three nitrogen atoms of BLM, is considered to be a valuable one for clinical use. This 99 m Tc species is chemically the most stable and a rapid clearance from blood is obtained; yielding a high tumor-blood ratio of 99 m Tc activity after injection into tumor-bearing mice. A selective labeling method for this 99 m Tc complex is described.

Isocitrate lyase from Pseudomonas indigofera III. Sulfhydryl groups and enzyme activity

1. The number of sulfhydryl groups per enzyme molecule was examined for various preparations of isocitrate lyase ( threo - D s -isocitrateglyoxalate-lyase, EC 4.1.3.1) from Pseudomonas indigofera by spectrophotometry using p -hydroxymercuribenzoate. It was: 9 for “active” enzyme, i.e. , enzyme for which EDTA could replace GSH in the assay; 4 for “inactive” enzyme prepared by treatment with GSSG, i.e. , enzyme for which EDTA could, not replace HSG; and 4–5 for either active or inactive enzyme pretreated with N -ethylmaleimide. Addition of 7–8 moles N -ethylmaleimide per mole active enzyme resulted in maximum inhibition. 2. Isocitrate plus Mg 2+ rather specifically protected enzyme against inactiva-tion by GSSG, when several combinations of substrates and cofactors (isocitrate, succinate, glyoxylate, Mg 2+ , and EDTA) were examined. Of several other organic compounds tested only malonate afforded some protection in the presence of Mg 2+ . Mn 2+ and Co 2+ replaced Mg 2+ to some degree in protection that was dependent upon the presence of isocitrate. 3. A possible method for selective labeling of enzymically essential SH by N -[ 14 C]ethylmaleimide was examined and appears promising.