S. L. Allman

The study of 2,3,4-trihydroxyacetophenone and 2,4,6-trihydroxyacetophenone as matrices for DNA detection in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric study of DNA detection using 2,3,4-trihydroxyacetophenone, 2,4,6-trihydroxyacetophenone, and their combination has been carried out systematically. The results show that a mixture of 2,4,6-trihydroxyacetophenone, 2,3,4-trihydroxyacetophenone and ammonium citrate with molar ratios of 2:1:1 serves as a good matrix for the detection of DNA, especially for samples containing a small quantity of DNA such as polymerase chain reaction product. The resolution and shot-to-shot reproducibility using this matrix are better than, and the MALDI sensitivity comparable to, that obtained when using 3-hydroxy picotinic acid (3-HPA), PA and ammonium citrate matrix (9:1:1). The mechanism of desorption/ionization using this matrix is discussed.

Caldicellulosiruptor obsidiansis sp. nov., an Anaerobic, Extremely Thermophilic, Cellulolytic Bacterium Isolated from Obsidian Pool, Yellowstone National Park

ABSTRACT A novel, obligately anaerobic, extremely thermophilic, cellulolytic bacterium, designated OB47T, was isolated from Obsidian Pool, Yellowstone National Park, WY. The isolate was a nonmotile, non-spore-forming, Gram-positive rod approximately 2 μm long by 0.2 μm wide and grew at temperatures between 55 and 85°C, with the optimum at 78°C. The pH range for growth was 6.0 to 8.0, with values of near 7.0 being optimal. Growth on cellobiose produced the fastest specific growth rate at 0.75 h−1. The organism also displayed fermentative growth on glucose, maltose, arabinose, fructose, starch, lactose, mannose, sucrose, galactose, xylose, arabinogalactan, Avicel, xylan, filter paper, processed cardboard, pectin, dilute acid-pretreated switchgrass, and Populus. OB47T was unable to grow on mannitol, fucose, lignin, Gelrite, acetate, glycerol, ribose, sorbitol, carboxymethylcellulose, and casein. Yeast extract stimulated growth, and thiosulfate, sulfate, nitrate, and sulfur were not reduced. Fermentation end products were mainly acetate, H2, and CO2, although lactate and ethanol were produced in 5-liter batch fermentations. The G+C content of the DNA was 35 mol%, and sequence analysis of the small subunit rRNA gene placed OB47T within the genus Caldicellulosiruptor. Based on its phylogenetic and phenotypic properties, the isolate is proposed to be designated Caldicellulosiruptor obsidiansis sp. nov. and OB47 is the type strain (ATCC BAA-2073).

Laser-induced acoustic desorption

Abstract The basic principles and experimental results of laser-induced acoustic desorption (LIAD) are reported in this work. LIAD was successfully used to desorb biomolecules in a mass spectrometer. LIAD holds some promise to eliminate many key factors which contribute to poor mass resolution in MALDI. Thus, it has potential for improvement in resolution of biomolecular mass analysis.

The Effect of Ammonium Salt and Matrix in the Detection of DNA by Matrix‐assisted Laser Desorption/Ionization Time‐of‐flight Mass Spectrometry

The effect of ammonium salt in the detection of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been systematically investigated using several matrices with ammonium salt additives. The results show that the presence of ammonium salt in the matrix has a beneficial effect on protonation and deprotonation of oligonucleotides in addition to suppressing alkali-ion adducts. These observations indicate that a good matrix for DNA detection could be made from two components; i.e. one component for desorption, which has a relatively large absorption coefficient at the incident laser wavelength, and another component for ionization, which has good protonation and deprotonation ability. Experimental results show that ammonium citrate and ammonium tartrate are good ionization components. Experimental results also indicate that large numbers of chemicals can be used as desorption components. The concept of a two-component matrix system opens many possibilities in the search for a good matrix to be used in the detection of biopolymers. The selection criteria for good matrices in this two-component system are discussed.

Method for counting noble gas atoms with isotopic selectivity

A method has been developed for direct counting of noble gas atoms and has been demonstrated for selected isotopes of krypton. In principle, a few atoms of the noble gases argon, krypton, xenon and radon can now be counted with isotopic selectivity whether stable or radioactive. A concept was originated in which a laser method would be used to count noble gas atoms of a particular isotope that are moving freely in an enclosure. As the concept developed, a parallel with Maxwells sorting demon became quite obvious since the plan was to sort out only atoms of a given type (Z selection), e.g. krypton atoms, from any other atom in the enclosure and then to sort the atom by isotope (A selection) before removing the atoms from the gas compartment. The plan was to count each atom as it was stored in a target until all atoms were counted.

Laser-induced acoustic desorption of electrons and ions

Laser irradiation of absorbing materials can be used to generate acoustic pulses with extremely high amplitude and short pulse duration. Such acoustic pulses can transfer energy and momentum to atomic particles on solid surfaces to cause desorption of the particles. We report experimental observations of the effect of laser-induced acoustic desorption (LIAD) of electrons from metal film surfaces and hydrogen ions from the surface of palladium saturated with hydrogen. We believe LIAD can be used as a gentle technique to transfer analyte molecules and ions into gas phase for mass analysis and for other applications.

Laser desorption mass spectrometry for point mutation detection

A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments generated by digestion of a PCR generated target fragment.

Oligonucleotide Sequencing by Fragmentation in Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Oligonucleotide sequencing by fragmentation in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has been demonstrated. With this sequencing method, the preparation of oligonucleotide ladder products and gel electrophoresis are not required, thus the sequencing speed potentially can be increased. Sequence information for single strand oligonucleotide segments up to 35 mer was obtained from fragmentation patterns using a mixture of anthranilic acid and ammonium citrate or 2,4,6-trihydroxyacetophenone and ammonium citrate as matrices. This method shows that the cleavage of P—O bond at 5′-linkage and C—O bond at 3′-linkage could be established in the MALDI process. Such cleavages at specific sites in the backbone of oligonucleotide can provide the foundation for fast oligonucleotide sequencing.

Miniature sensor suitable for electronic nose applications

A major research effort has been devoted over the years for the development of chemical sensors for the detection of chemical and explosive vapors. However, the deployment of such chemical sensors will require the use of multiple sensors (probably tens of sensors) in a sensor package to achieve selective detection. In order to keep the overall detector unit small, miniature sensors with sufficient sensitivity of detection will be needed. We report sensitive detection of dimethyl methylphosphonate (DMMP), a stimulant for the nerve agents, using a miniature sensor unit based on piezoresistive microcantilevers. The sensor can detect parts-per-trillion concentrations of DMMP within 10 s exposure times. The small size of the sensor makes it ideally suited for electronic nose applications.

Matrix‐assisted laser desorption/ionization for short tandem repeat loci

Matrix-assisted laser desorption/ionization was used for the detection of four base short tandem repeats (STR) for clinical samples using a time-of-flight mass spectrometer. Since STR plays an important role in genetic disease and human identification, this work indicates that laser desorption mass spectrometry has the potential to achieve rapid DNA typing for both forensic applications and genetic disease diagnosis.