Sugar additives for MALDI matrices improve signal allowing the smallest nucleotide change (A:T) in a DNA sequence to be resolved

Sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) of DNA is critical for obtaining high quality mass spectra. Sample impurity, solvent content, substrate surface and environmental conditions (temperature and humidity) all affect the rate of matrix–analyte co-crystallization. As a result, laser fluence threshold for desorption/ionization varies from spot to spot. When using 3-hydroxypicolinic acid (3-HPA) as the matrix, laser fluence higher than the threshold value reduces mass resolution in time-of-flight (TOF) MS as the excess energy transferred to DNA causes metastable decay. This can be overcome by either searching for ‘hot’ spots or adjusting the laser fluence. However, both solutions may require a significant amount of operator manipulation and are not ideal for automatic measurements. We have added various sugars for crystallization with the matrix to minimize the transfer of excess laser energy to DNA molecules. Fructose and fucose were found to be the most effective matrix additives. Using these additives, mass resolution for DNA molecules does not show noticeable deterioration as laser energy increases. Improved sample preparation is important for the detection of single nucleotide polymorphisms (SNPs) using primer extension with a single nucleotide. During automatic data acquisition it is difficult to routinely detect heterozygous A/T mutations, which requires resolving a mass difference of 9 Da, unless a sugar is added during crystallization

Specific recognition of non-denatured nitrite-oxidizing system ofNitrospira moscoviensis by monoclonal antibody Hyb 153-3

The objective of this research project is to develop a rapid molecular method for monitoring nitrification in a wastewater reactor. In the developed method, a monoclonal antibody (Hyb 153-3) was used because it can specifically recognize non-denatured enzymes responsible for nitrite oxidation in Nitrobacter and Nitrospira. The recognition of these enzymes under non-denatured conditions can significantly simplify the procedures of future immunoassays for environmental samples collected from various natural and engineered systems. This paper presents the ability of the selected Hyb 153-3 antibody to recognize the non-denatured form of the nitrite-oxidizing enzyme of Nitrospira in an aqueous phase as well as when the enzyme has been immobilized on a solid surface. Copyright © 2005 Society of Chemical Industr

Investigation of Enzymatic Behavior of Benzonase/Alkaline Phosphatase in the Digestion of Oligonucleotides and DNA by ESI-LC/MS

We have developed an ion-pairing HPLC-MS method that has sufficient separation power, selectivity, and sensitivity to investigate the enzymatic behavior of benzonase/alkaline phosphatase upon digestion of oligonucleotides and DNA. Mass spectrometry revealed that this enzyme pair can nonspecifically digest oligonucleotides and DNA into fragments ranging from 2 to 10 nucleotides, i.e., sizes suitable for routine mass spectrometric measurements. Trimers, tetramers, and pentamers are the most prominent digested products. This makes benzonase/alkaline phosphatase a promising choice for DNA and DNA adduct related studies that require a nonspecific enzyme. A computer software program developed in-house was critical in automating the processing of mass spectral data. The methodology described here provides a systematic approach for evaluating the behavior of DNA-cleaving enzymes by mass spectrometry

Base-specific fragmentation of amplified 16S rRNA genes and mass spectrometry analysis: A novel tool for rapid bacterial identification.

A rapid approach to the 16S rRNA gene (16S rDNA)-based bacterial identification has been developed that combines uracil-DNA-glycosylase (UDG)-mediated base-specific fragmentation of PCR products with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). 16S rDNA signature sequences were PCR-amplified from both cultured and as-yet-uncultured bacteria in the presence of dUTP instead of dTTP. These PCR products then were immobilized onto a streptavidin-coated solid support to selectively generate either sense or antisense templates. Single-stranded amplicons were subsequently treated with uracil-DNA-glycosylase to generate T-specific abasic sites and fragmented by alkaline treatment. The resulting fragment patterns were analyzed by MALDI-TOF MS. Mass signals of 16S rDNA fragments were compared with patterns calculated from published 16S rDNA sequences. MS of base-specific fragments of amplified 16S rDNA allows reliable discrimination of sequences differing by only one nucleotide. This approach is fast and has the potential for high-throughput identification as required in clinical, pharmaceutical, or environmental microbiology. In contrast to identification by MS of intact whole bacterial cells, this technique allows for the characterization of both cultured and as-yet-uncultured bacteria

A simple approach for evaluating total MicroRNA extraction from mouse brain tissues

For the analysis of microRNA, a common approach is to first extract microRNA from cellular samples prior to any specific microRNA detection. Thus, it is important to determine the quality and yield of extracted microRNA. In this study, solid-phase extraction was used to isolate small RNA (<200 nt), which included microRNA, from mouse brain tissues. By using standard UV absorbance measurements, the amount of small RNA in the extracted RNA samples was determined. To determine the presence of microRNA, each RNA sample was analyzed by PAGE with SYBR[R] Green II staining. Testing for contamination of any small DNA fragments, RNase and cellular peptides or proteins were systematically carried out. By scanning the gel image obtained from PAGE analysis, the average percentage of total microRNA (19-25 nt) in the extracted RNA samples was determined to be equal to 2.3% [+ or -] 0.5%. The yield of total microRNA was calculated to be ~0.5 ng of microRNA per milligram of frozen mouse brain tissue. In comparison to other methods that require the use of expensive specialized instrumentation, the approach of combining the standard UV absorbance and PAGE analysis represents a simple and viable method for evaluating the quality and yield of microRNA extraction from tissue samples

Comparison of Accuracy on DNA Quantitation Determined by MALDI-TOF Mass Spectrometry and UV Spectrometry

Although the UV absorbance of DNA at 260 nm has been recognized as a standard method for DNA quantitation, there are limitations of using UV spectrometry to determine the purity and identity of DNA. Recently, MALDI-TOF MS has proven to be an accurate technique for qualitative DNA analysis. In this study, the accuracy of MALDI-TOF MS for determining the concentration of DNA is evaluated and compared with that of the standard UV method. The results indicated that the accuracy of quantitative MALDI-TOF MS was comparable to that of the standard UV method and that measured DNA concentrations correlated well with those determined by the standard UV method

Heterobifunctional linker between antibodies and reporter genes for immunoassay development

The amplification inherent in transcription and translation of DNA has already been exploited for the development of highly sensitive immunoassays by using a reporter gene as a label that, upon in vitro expression, generates multiple enzyme molecules in solution (expression immunoassay). The most challenging task in the development of an expression immunoassay is to link the antibody to a reporter gene that also contains control elements for transcription/translation. In this work, we prepare heterobifunctional linkers that consist of a modified avidin or streptavidin covalently attached to an oligonucleotide (dA)40. (Strept)avidin interacts with a biotinylated detection antibody whereas the oligonucleotide hybridizes with a complementary poly(dT) tail added enzymically to the 3' end of the reporter gene. The linker is evaluated in a model two-site (sandwich-type) immunoassay performed in microtiter wells. A 4.3 kb plasmid containing the firefly luciferase cDNA is used as a reporter

Comparison of Fluorometric Detection Methods for Quantitative Polymerase Chain Reaction (PCR)

In this study, we compared the sensitivity of two different detection methods for quantitative polymerase chain reaction (PCR). Various amounts of a 75 mer single-stranded deoxyribonucleic acid (DNA) fragment, which can be used as a DNA label for the immuno-PCR (iPCR) assays, were amplified by PCR. The amount of amplified DNA fragments was determined by the fluorescence (FL) of SYBR(R) Green dye that specifically interacts with double-stranded DNA fragments. In the first selected detection method, real-time PCR, FL measurements were carried out at each thermal cycle, as the DNA was being amplified by PCR. This was achieved using the Applied Biosystems (ABI) Prism 7000 Sequence Detection System and its standard protocol. In the second detection method, referred to as end-point detection, after the PCR amplification was completed, off-line FL measurements were subsequently carried out using a conventional plate reader. In order to achieve the lowest limit of detection (LOD) from the off-line measurement, we have optimized a wide variety of parameters. Our data have indicated the LOD of real-time PCR method was approximately three orders of magnitude lower than the end-point measurement method, with a linear range spanning six orders of magnitude; 10 fmol to 10 zmol of PCR template. The lower LOD of the real-time PCR method could be partly due to the ability to maximize the number of thermal cycles that could be carried out in PCR, without increasing the nonspecific amplification of any contaminating DNA. The results of this study can be applied to the development of ultra-sensitive iPCR assays for various disease markers

Expression Immunoassay. Antigen quantitation using antibodies labeled with enzyme-coding DNA fragment.

A novel immunoassay is reported which uses an enzymecoding DNA fragment as label (expression immunoassay). The DNA label is determined with high sensitivity by measuring the enzymatic activity produced after expression. A DNA fragment encoding the firefly luciferase is biotinylated and complexed with streptavidin. Biotinylated, specific antibodies are used for quantitation of antigen immobilized on microtiter wells. After completion of the immunoreaction, streptavidin-DNA is bound to the immunocomplex. Subsequent expression of the solid phase-bound DNA, by an one-step (coupled) cell-free transcription/translation, produces luciferase. The enzyme catalyzes the luminescent reaction of luciferin with 02 and ATP. As few as 3000 molecules of DNA label can be detected. Also, 50000 antigen molecules can be detected, and the luminescence is a linear function of the number of antigen molecules in a range extending over 3 orders of magnitude. The high sensitivity achieved is a result of the combined amplification due to transcription/ translation and the substrate turnover

Reduction of Internal Standard Signals in Quantitative MALDI-TOF Mass Spectrometry

The advantages of combining qualitative and quantitative analysis on a single analytical technique have further extended the applications of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to the quantitation of various biomolecules. To achieve absolute quantitation, it is necessary to perform a calibration with standard dilutions. For the purpose of measuring DNA samples, a pure DNA oligonucleotide at different concentrations was chosen as a standard to perform the calibration of MALDI-TOF MS. In order to overcome the variation of signal intensity from repeated measurements of each DNA standard dilution, fixed amount of an internal standard was added into each DNA standard dilution. Instead of maintaining at a constant level, the signals of fixed amount of internal standard were decreased 73% from its initial level while the signals of DNA standard continued to increase within a linear dynamic range for quantitation from 0.20 µM to 12.5 µM of DNA. Attempts to identify the cause of signal reduction were systematically carried out. This is the first report on the extent of signal reduction in quantitative MALDI-TOF MS. These results represent a limitation on using MALDI-TOF MS to monitor the changes in concentration of two different compounds within a chemical or biological system