N. I. Taranenko

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.

SURFACE-ENHANCED RAMAN DETECTION OF NERVE AGENT SIMULANT (DMMP AND DIMP) VAPOR ON ELECTROCHEMICALLY PREPARED SILVER OXIDE SUBSTRATES

Surface-enhanced Raman scattering (SERS) spectra of the chemical nerve agent simulants dimethyl methylphosphonate (DMMP) and diisopropyl methylphosphonate (DIMP) were obtained in the vapor phase. The spectra obtained demonstrate the possibility of detecting these nerve agent species in the vapor phase using electrochemically roughened silver oxide substrates prepared from silver foil.

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.

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.

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.

Detection of trinucleotide expansion in neurodegenerative disease by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Genotyping of the dentatorubral-pallidoluysian atrophy (DRPLA) locus in six patient samples, representing four normal individuals and two DRPLA patients, was successfully obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). DRPLA is a dominantly inherited neurodegenerative disorder associated with the expansion of an unstable trinucleotide (CAG) repeat. The accurate determination of repeat length utilizing MALDI supports the use of this methodology for the analysis of genes containing unstable CAG trinucleotide repeats.

Matrix-assisted Laser Desorption/Ionization for Sequencing Single-stranded and Double-stranded DNA

The DNA sequence of a single-stranded and double-stranded template was determined. The templates were sequenced using the chain termination method and cycle sequencing method and detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The sequencing products were analyzed successfully without the laborious and expensive methods for removal of the template. Direct sequencing of the double-stranded template was achieved with minimal post-reaction purifications, which could be extremely important for mutation analysis and clinical diagnosis. A systematic study of the mechanisms and kinetics of sequencing reactions was also performed. The details of this analysis and directions for future improvements of the quality of sequencing are presented.

DIRECT MONITORING OF LASER ABSORPTION OF MALDI MATRICES BY FAST PIEZOELECTRIC TRANSDUCER

Abstract The absorption of laser energy by recently found matrices used in matrix assisted laser desorption ionization (MALDI) detection of large DNA segments: 3-hydroxypicolinic acid (3-HPA), picolinic acid (PA), composite matrix (PA + 3-HPA) and 3-aminopicolinic acid (3-APA), was studied by using a fast piezoelectric transducer for solid matrices and for their aqueous solutions. A nonlinear dependence of acoustical signal on laser fluence was found for PA, PA + 3-HPA and 3-APA matrices at 266 nm. Near linear dependence was found for 3-HPA at 266 nm. There was no strong deviation from linear power dependence for any of these matrices at 355 nm. Possible mechanisms to account for the observed nonlinearity are discussed.

Fast DNA analysis by laser mass spectrometry for human genome analysis

Fast DNA sequencing by laser mass spectrometry is possible if the following 3 criteria are met: (1) Size of DNA fragment should be greater than 300 nucleotides. (2) Enough sensitivity to detect DNA produce from polymerases chain reactins (PCR). (3) Higher resolution of mass spectr. So far, the firt 2 criteria are met: If the resolution can be significantly improve, fast DNA sequencing by laser mass spectrometry weil be a reality in the near feature. (AIP)