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Miyazaki K, Tsugita A. C-terminal sequencing method for proteins in polyacrylamide gel by reaction of acetic anhydride. Proteomics 6;2006:2026–2033. [PubMed]
A reaction scheme for removing amino acids from the C-termini of polypeptides is adapted for use on proteins in polyacrylamide gels to deduce C-terminal sequence information. The chemistry relies on the use of acetic anhydride to form C-terminal oxazolones. Gel pieces are first dehydrated, then immersed in acetic anhydride in formaldehyde solution. The truncation reaction is allowed to proceed at 50°–60°C for several hours. The gel is washed in aqueous dimethylaminoethanol, and finally incubated in that reagent at 60°C for 1 h to open C-terminal oxazolone rings and hydrolyze O-acetylated residues. The resulting truncated protein species are digested with trypsin, and MALDI-TOF mass spectrometry is employed to study the C-terminal truncated fragments corresponding to the C-terminal amino acid sequence of the protein. The negative-ion mode is used to enhance relative signal strength of the C-terminal ions of interest. The sensitivity of the method enables C-terminal sequences to be deduced from low-microgram quantities of protein.
Zhang K, Zhu J, Shendure J, Porreca GJ, Aach JD, Mitra RD, Church GM. Long-range polony haplotyping of individual human chromosome molecules. Nature Genetics 38;2006:382–387. [PubMed]
This work provides rapid methodology for molecular haplotyping over distances greater than 100 kb. The method is based on the localized, clonal amplification of DNA template molecules immobilized in a thin layer of polyacrylamide gel on a microscope slide and then interrogated in parallel by in situ probing or sequencing with the use of an imaging technique. The methodology is here applied to individual chromosome molecules. Genotypes are determined in situ by multiple rounds of single base extension assays. Haplotypes spanning a 153-Mb region of human chromosome 7 are determined. Evidence for rare mitotic recombination events in human lymphocyes is presented. Moreover, by pooling DNA from a population of individuals on one slide, very high throughput haplotyping is achievable, and the linkage disequilibrium information acquired is more accurate than that based on statistically inferred haplotypes.
Tabone T, Sallmann G, Webb E, Cotton RGH. Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: A novel concept for mutation scanning. Nucleic Acids Research 34;2006:e45. [PubMed]
A simple optical absorbance assay is shown to be capable of detecting DNA polymorphisms and mutations in amplicons made from mammalian DNA samples. The basis of the method is detection of oxidation products of mismatched thymine and cytosine bases by potassium permanganate as it is reduced to manganese dioxide. The method efficiently detects insertions, deletions, and nucleotide substitutions in heterodupex DNA, and does so without requiring fluorescent primers, labeled probes, or electrophoretic separations. The assay has a single universal protocol that does not require optimization for each new amplicon to be analyzed.
Hu Z, Zhang A, Storz G, Gottesman S, Leppla SH. An antibody-based microarray assay for small RNA detection. Nucleic Acids Research 34;2006:e52. [PubMed]
This paper presents a sensitive method for detecting RNAs hybridized to DNA microarrays that is based on the use of an antibody that recognizes RNA/DNA hybrids with high affinity in a sequence-independent manner. The antibody, S9.6, is derived from a mouse hybridoma cell line available from ATCC. Its binding to microarrays is detected with a biotin-labeled anti-IgG and fluorescently labeled streptavidin. The method is sufficiently sensitive to detect binding by structured and small RNA species. It will be of interest to investigators wishing to detect DNA/RNA hybrids on spotted microarrays.
Cai S-S, Syage JA. Comparison of atmospheric pressure photoionization, atmospheric pressure chemical ionization, and electrospray ionization mass spectrometry for analysis of lipids. Analytical Chemistry 78;2006:1191–1199. [PubMed]
This work evaluates the sensitivity and quantitative accuracy of lipid analysis using three widely available mass spectrometric ionization techniques: electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI). Neutral lipids are selected for analysis, including free fatty acids, and their monoglyceride, diglyceride, and triglyceride esters. Normal-phase chromatography is performed for on-line separation of these non-polar compounds. APPI is found to provide the best limits of detection, the highest signal intensities, and highest signal-to-noise ratios. APPI and APCI provide comparable dynamic range of four to five orders of magnitude. ESI sensitivity is greatly enhanced by addition of mobile phase modifiers, but the linear range of quantitation for the resulting adduct species is curtailed.
Jones JJ, Borgmann S, Wilkins CL. Characterizing the phospholipids profiles in mammalian tissues by MALDI FTMS. Analytical Chemistry 78;2006:3062–3071. [PubMed]
Mass spectrometric profiling of tissue phospholipids is accomplished with a sample preparation method consisting simply of sonicating tissue in a solution of 2,5-dihydroxybenzoic acid as MALDI matrix. The sonicated tissue samples are then spotted directly onto target plates. Mass analysis of the resulting complex phospholipids mixtures is performed by FTMS to take advantage of the high mass accuracy and resolution available. Measurements are performed at a resolving power of 12,000, enabling resolution of multiple ions having the same nominal m/z value.
Edelson-Averbukh M, Pipkorn R, Lehmann WD. Phosphate group–driven fragmentation of multiply charged phosphopeptide anions. Improved recognition of peptides phosphorylated at serine, threonine, or tyrosine by negative ion electrospray tandem mass spectrometry. Analytical Chemistry 78;2006:1249–1256. [PubMed]
A well-established method for phosphopeptide detection is precursor ion scanning in the negative ion mode, in which the inorganic marker ion, [PO3]−, is monitored at m/z 79. The present publication shows that multiply charged precursor phosphopeptide anions fragment to yield products of the form [M − nH − 79](n − 1)−, where n = the number of charges. The precursor ions are proposed to carry two negative charges on the phosphate group. One negative charge is lost with the loss of each phosphate. Phosphopeptides can therefore be recognized by scanning for precursors of the [M − nH − 79](n − 1)− product ions. This method will be more sensitive than monitoring the inorganic marker ion on some instruments.
Tagwerker C, Flick K, Cui M, Guerrero C, Dou Y, Auer B, Baldi P, Huang L, Kaiser P. A tandem affinity tag for two-step purification under fully denaturing conditions. Application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking. Molecular and Cellular Proteomics 5;2006:737–748. [PubMed]
The method described in this paper for purification of proteins through affinity tags combines the high degree of purity attainable with tandem tagging and advantages of purification under denaturing conditions. Specifically, denaturing conditions are used in the present report to suppress reactions catalyzed by endogenous enzymes in cell extracts that remove post-translational modifications of interest from proteins under study. The two tags employed are: a hexahistidine tag for purification by nickel chelation affinity chromatography, and a 75-amino-acid sequence that is efficiently biotinylated in yeast and mammalian cells for purification by streptavidin affinity chromatography. Cells are lysed in 8 M urea or 6 M guanidinine hydrochloride, and subsequent tandem affinity purification maintains these denaturing conditions throughout. The biotin tag may be removed subsequently by inclusion of a TEV protease cleavage site.
Khan F, He M, Taussig MJ. Double-hexahistidine tag with high-affinity binding for protein immobilization, purification, and detection on Ni-nitrilotriacetic acid surfaces. Analytical Chemistry 78;2006:3072–3079. [PubMed]
A double hexahistidine tag is used to increase the affinity of binding to Ni-NTA for purification of tagged proteins. The two hexahistidine units are separated by an 11-amino-acid spacer. This provides 10-fold higher affinity than either a single hexahistidine tag or two separate hexahistidine tags with one placed on the protein N-terminus and the other on the C-terminus. The new tag will be useful both for protein purification and for immobilizing proteins to Ni-NTA chips for Biacore surface plasmon resonance studies.
Tang Y, Mernaugh R, Zeng X. Nonregeneration protocol for surface plasmon resonance: Study of high-affinity interaction with high-density biosensors. Analytical Chemistry 78;2006:1841–1848. [PubMed]
Karlsson R, Katsamba PS, Nordin H, Pol E, Myszka DG. Analyzing a kinetic titration series using affinity biosensors. Analytical Biochemistry 349;2006:136–147. [PubMed]
Conventionally, protocols in which surface plasmon resonance is used to measure kinetic and thermodynamic constants for molecular interactions involve the need to remove surface-bound analyte from complexes to regenerate the immobilized ligand between successive analyte injections. This procedure is commonly problematic because the sometimes harsh conditions that may be needed to disrupt complexes may also degrade the binding activity of the ligand. The present articles describe protocols in which regeneration is not required. Instead, analyte is injected in steps of increasing concentration to produce successively higher levels of surface binding, and association and dissociation curves are acquired at each step. A relatively high density of immobilized ligand is required for this analysis to acquire enough data points before surface saturation is reached. This requirement is facilitated by use of ligands of small size. The method will be useful for measuring interactions of very high affinity where regeneration is especially difficult.
Meiring HD, Soethout EC, Poelen MCM, Mooibroek D, Hoogerbrugge R, Timmermans H, Boog CJ, Heck AJR, de Jong APJM, van Els CACM. Stable isotope tagging of epitopes. Molecular and Cellular Proteomics 5;2006:902–912. [PubMed]
MHC Class I–associated peptides that are expressed in cells specifically in the course of viral infection are identified with the help of a stable isotope labeling strategy. Virus-infected cells are cultured in the presence of isotopically labeled amino acids. These cells are then mixed with uninfected cell cultures in normal medium. MHC molecules are purified immunochemically and associated peptides are eluted in acid for LC/MS analysis. Self-peptides display isotopic distributions characteristic of the admixture. However, viral peptides are produced exclusively in the virus-infected cells and have no counterpart in the uninfected cells. They therefore have a correspondingly different isotopic distribution that can be detected by automated means. Using this method, novel measles virus and respiratory syncitial virus peptides are detected, as well as infection-induced self-peptides.
Klammer AA, MacCross MJ. Effects of modified digestion schemes on the identification of proteins from complex mixtures. Journal of Proteome Research 5;2006:695–700. [PubMed]
A variety of trypsin digestion protocols for use in identifying cell lysate proteins by LC/MS/MS are compared with respect to the number of proteins identified. Among the findings is that digestion in the presence of the acid-cleavable detergent RapiGest (Waters, Milford, MA), produces increased numbers of assignments compared with urea. Secondly, shorter digestion times work as well as or better than longer times. Thirdly, digestions performed by pumping the protein mixture through a column of immobilized trypsin produce more assignments than digestion in tubes. The results indicate that relatively subtle changes in digestion methods can have a strongly positive effect on the outcome of proteomic experiments.
Wyttenbach A, Tolkovsky AM. Differential phosphorylation labeling (DIPPL), a method for comparing live cell phosphoproteomes using simultaneous analysis of 33P- and 32P-labeled proteins. Molecular and Cellular Proteomics 5;2006:553–559. [PubMed]
The phopshoproteomes of two cell lines or tissues are compared by labeling one with 32P and the other with 33P, mixing proteins from the two cell lines/tissues for co-electrophoresis, and then detecting phosphoproteins by autoradiagraphy. The emission energy for 33P is sevenfold lower than 32P. Two exposures are performed. The first detects both 32P- and 33P-labeled proteins. The second is performed with two acetate sheets between the gel and the detection plate, to screen out emissions from 33P. This method is applied to the detection of kinase substrates by labeling with 32P cells in which the kinase of interest is inactive, and labeling with 33P cells in which the kinase is active. The method is very sensitive, and is suitable for study of tissues available only in low amounts, such as primary neurons.
Schindler J, Lewandrowski U, Sickmann A, Friauf E, Nothwang HG. Proteomic analysis of brain plasma membranes isolated by affinity two-phase partitioning. Molecular and Cellular Proteomics 5;2006:390–400. [PubMed]
This paper provides a subcellular fractionation method for enrichment of brain plasma membrane proteins. The method is based on partition of microsomes in an aqueous two-phase system formed when solutions of polyethylene glycol (PEG) and dextran are mixed. Microsomes derived from different subcellular compartments distribute between the phases on the basis of differing membrane surface properties, such as charge and hydrophobicity. Plasma membrane microsomes have a higher affinity for the PEG-enriched phase. They are then further enriched by affinity for wheat-germ agglutinin coupled to dextran, to which they bind through their content of the plasma membrane markers N-acetyl-D-glucosamine and sialic acid. The proteins present in the microsomes purified in this protcol are then available for proteomic characterization. The method is suitable for use with small amounts of brain tissue, such as the cerebellum of a single rat, is completed in 4 h, and maintains protein interactions so that multisubunit complexes may be analyzed.
Ruth MC, Old WM, Emrick MA, Meyer-Arendt K, Aveline-Wolf LD, Pierce KG, Mendoza AM, Sevinsky JR, Hamady M, Knight RD, Resing KA, Ahn NG. Analysis of proteins from human chronic myelogenous leukemia cells: Comparison of extraction methods for multidimensional LC-MS/MS. Journal of Proteome Research 5;2006:709–719. [PubMed]
Four different methods for extracting membrane proteins for trypsin digestion and LC/MS/MS are compared with respect to their efficiency of protein solubilization and recovery, their enrichment of membrane proteins, and the peptide coverage of transmembrane proteins they support. Three of the methods have been previously published. They are extraction in acid-labile surfactant, methanol, and urea (followed by dilution to trypsin-compatible urea concentrations). The fourth method also uses urea extraction, but adds a rapid desalting step instead of dilution. Extraction with acid-labile surfactant, urea followed by dilution, and urea followed by desalting, all provided comparable peptide recoveries and sequence coverage of transmembrane proteins. However, poorer proteolysis is seen with organic solvent.
Yen C-Y, Russell S, Mendoza AM, Meyer-Arendt K, Sun S, Cios KJ, Ahn NG, Resing KA. Improving sensitivity in shotgun proteomics using a peptide-centric database with reduced complexity: Protease cleavage and SCX elution rules from data mining of MS/MS spectra. Analytical Chemistry 78;2006:1071–1084. [PubMed]
In order to improve discrimination between correct and incorrect peptide assignments during automated database searching, procedures for editing peptide sequence databases are developed in this paper to permit smaller databases to be searched and hence false-positive assignments to be minimized. By inspecting a set of high-confidence assignments, rules for unlikely missed cleavages and non-tryptic proteolysis products are developed. The elution behavior of peptides from ion-exchange columns is also employed to edit the peptide database searched. Decreased numbers of incorrect assignments with scores higher than correct peptide sequences are observed. Additionally, a major class of common, non-specific proteolysis products is identified as corresponding to leucine aminopeptidase cleavage. This insight enables substantial improvement to be made in the identification of non-tryptic peptides through appropriate peptide database editing. The results provide better confidence than indiscriminate search routines based on unspecified enzyme cleavage.
Nettikadan S, Radke K, Johnson J, Xu J, Lynch M, Mosher C, Henderson E. Detection and quantification of protein biomarkers from fewer than 10 cells. Molecular and Cellular Proteomics 5;2006:895–901. [PubMed]
This paper illustrates the gains in sensitivity to be made by miniaturizing microarray formats. Antibody microarrays are constructed with spot diameters in the range 1–20 μm. The associated high spot densities and correspondingly low surface area for the array permit analysis of very small sample volumes (1–6 μL in this study, although the arrays are compatible with submicroliter volumes). Arrays for the detection of interleukin-6 and prostate-specific antigen (PSA) are tested, and are shown to provide detection sensitivities in the attamole range. Cellular PSA is detected from the lysate of an average of just six cells.
Ngo VN, Davis RE, Lamy L, Yu X, Zhao H, Lenz G, Lam LT, Dave S, Yang L, Powell J, Staudt LM. A loss-of-function RNA interference screen for molecular targets in cancer. Nature 441;2006:106–110. [PubMed]
RNA interference (RNAi) is used in a genetic screen for key regulators of cancer cell proliferation and survival. Interfering RNAs that target such genes are expected to be toxic. Therefore, small hairpin RNAs (shRNAs) are introduced in an inducible retroviral expression vector. When induced, target gene products are knocked down by 50–70%. A library of shRNAs targeting 2500 human genes is constructed, with three to six shRNAs per gene. Each shRNA construct bears a different 60-bp bar-code tag that is used to monitor the abundance of each shRNA in a cell culture. Any shRNA that knocks down the product of a gene essential for proliferation or survival is selectively eliminated from the culture. This is detected by amplifying the bar-code sequences in genomic DNA and co-hybridizing with control DNA from uninduced cells to a DNA microarray. In this way, the depletion of the bar-code DNA in cells expressing a given shRNA is quantitated. This experimental platform differs from previous RNAi screens for cancer genes, which rely on the rescue of cells from a toxic or cytostatic effect. These screens readily detect tumor-suppressor genes, but are not well suited for detection of oncogenes that promote the malignant phenotype. In the present method, interfering RNAs produce a cytotoxic or cytostatic effect, and thus reveal oncogenes that promote malignancy. It is hoped that the methodology will result in the creation of a molecular taxonomy of cancers based on which regulatory proteins or pathways promote cancer cell survival.
Schattner P, Diekhans M. Regions of extreme synonymous codon selection in mammalian genes. Nucleic Acids Research 34;2006:1700–1710. [PubMed]
While non-synonymous mutations (i.e., mutations resulting in amino acid substitution) have long been recognized to be subject to evolutionary selective pressure, evidence is now accumulating to indicate that synonymous mutations too may not be selectively neutral. Selection is attributed to cis-regulatory motifs, exonic splicing enhancers (ESEs), and mRNA secondary structures. In the present paper, a program is described for identifying such elements based on extreme codon conservation between homologous genes in related species. The program is applied to five mammalian species, and over 200 regions of extreme codon conservation are identified, ranging from 60 to 178 codons in length. They are located in genes involved in DNA-binding, RNA-binding, or binding to zinc ions. 43% of them overlap conserved alternative transcript isoforms and are enriched in ESE motifs. Others may function in mRNA stabilization or degradation. The methodology is anticipated to be a helpful tool for discovery of further mRNA processing motifs.