Probing for mutations
biotinylated DNA on a magnetic bead with streptavidin, hybridized PNA probes to the immobilized DNA washed away any unhybridized PNA and directly analyzed the The hunt has been on for the past decade remaining PNA by MALDI-TOF (time-ofto find methods that can be used for the analysis of DNA variations. A recent contri- flight) MS. Because of the strong biotinbution from Lloyd M. Smith and his co-work- streptavidin bond, the PNA can be dissociated without removing its DNA ers at the University of Wisconsin-Madison is an assay using peptide nucleic acid (PNA) complement. "affinity" MALDI (matrix-assisted laser deThey investigated as a model system sorption/ionization) MS. They immobilized the 182 base-pair gene that codes for tyrosinase, an enzyme in the melanin biosynthetic pathway. Mutations in the tyrosinase gene have been implicated in a type of albinism. They designed two PNA probes, one complementary to the wild-type DNA and one complementary to the mutation, for each of the four polymorphic sites in the gene. The probes were each mass-labeled with different numbers of 8-amino-3,6-dioxaoctanoic acid molecules to provide a distinct, singly charged molecular ion peak. With the method, they were able not only to identify multiple polymorphic sites but also to unambiguously identify heterozygotes. Three of the original probes gave satisfactory signal intensities and specificities, but the fourth probe had to be redesigned with a higher percentage of guanine and cytosine. One limitation of the assay is the requirement for different wash conditions for each of the probes to provide good discrimination between the wild-type and variant Schematic of the assay, showing biotinylprobes. The authors tried to combine the ated PCR products being attached to beads from separate reaction tubes in a magnetic beads; the nonbiotinylated strand single analysis, but finding conditions in being washed away; and the PNA probe which all the probes showed satisfactory being hybridized, washed, and analyzed by signal intensity proved difficult. {Nature MALDI. (Adapted with permission. Copyright Biotecknol. 1197,15,1368-72) 1997 Nature America.)
Integrating electrochemistry and biotechnology Bioelectrosynthesis—the combination of electrochemistry and enzyme catalysis— could be a promising way to drive certain reactions to high yields while avoiding toxic chemicals. For this technology to become practical, electrolysis must occur at an acceptable current density. Philip N. Bartlett and colleagues at The University of Southampton (U.K.) investigated increasing the current density at enzyme-modified electrades. The enzyme was immobilized on either a cylindrical reticulated vitreous carbon electrode or a vitreous carbon disk electrode.
In this study, the enzyme horseradish peroxidase was adsorbed onto a poly (aniline)-coated carbon surface and immobilized with a poly(l,2-diaminobenzene) film. The reduction of 2.4-mM hydrogen peroxide was employed as a model reaction. The electrodes operated under partial mass transport control and gave a current of —40 mA/cm3. The authors found that the current density depended on the surface loading of the enzyme and the kinetics of substrate conversion by the enzyme In addition, the lifetime of the electrode could be increased by crosslinking the enzyme with glutaraldehyde. The crosslinked electrodes did not lose activity, even after more than one day of electrolysis. (J. Electrochem. .oc. 1997, 44, 3705-10)
MALDI gets the (Coomassie) Blues Proteins are often separated in Coomassie Blue-stained sodium dodecyl sulfate-polyacrylamide gels. MALDI (matrix-assisted laser desorption/ ionization) MS would be a powerful method for the characterization of these gel-separated proteins. Unfortunately, the Coomassie Blue causes problems for mass spectrometric detection, primarily because of the formation of adducts. Hanno Ehring and co-workers at Pharmacia & Upjohn (Sweden) reported a new strategy for the MALDI MS analysis of proteins separated in Coomassie Blue-stained gels. They extracted the proteins directly from the gel with a mixture of formic acid, acetonitrile, isopropanol, and water in an ultrasonic bath. Part of the supernatant was then mixed with the matrix solution (2,5dihydroxybenzoic acid (DHB), 2-hydroxy-5-methoxybenzoic acid, or a-cyano4-hydroxycinnamic acid) for MALDI MS analysis. The remainder of the supernatant was dried to remove the formic acid and then digested with trypsin. Using DHBs as the matrix provided the best mass accuracy and sensitivity. Undiluted analyte, still containing the extraction solvent mixture, could be mixed with the matrix solution without compromising crystallization. The mass accuracy of the analyte spectra was not as good as that of the standards, with the masses shifted to higher values than expected. The authors speculate that this may be caused by the formylation of serine and threonine residues or by electrophoresisinduced modifications. The method was found to be applicable to proteins as large as 30 kDa. However, problems with sensitivity and resolution degradation were noted, especially when small amounts of proteins with molecular weights greater than 20 kDa were loaded on the gel. (Rapid Cotnmun. Mass Spectrom. 1997,71, 1867-73)
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