We report the first detection of DNA segments as large as 500 nucleotides by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, using picolinic acid and 3-hydroxypicolinic acid mixtures as desorption matrices. The successful detection of 500-nucleotide DNA indicates that laser mass spectrometry is now emerging as a new biotechnology tool for DNA-related research. It should be possible to utilize fast detection of large DNA segments by laser mass spectrometry for rapid human genome sequencing.
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.
The effect of ammonium salt in the detection of oligonucleotides by matrix-assisted laser desorptionlionization 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 coeflicient 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 twocomponent matrix system opens many passibilities 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.The development of oligonucleotide detection using Matrixassisted laser desorption/ionization (MALDI) mass spectrometry has received great attention during the past several years due to its potential application in DNA sequencing. Tremendous effort has been put into the improvement of MALDI instrumentation and the search for more effective matrices. The former has led to several new techniques in M U D 1 instrumentation."* However, the results of extensive searches for new matrices, which are suitable for the detection of oligonucleotides, have fallen short of expectations. Good matrices have been limited to a few molecules, such as 3-hydroxypicolinic acid3' (3-HPA), picolinic acid4 (PA), and trihydroxyacetophenone? This slow progress in the search for effective matrices perhaps relates to the concept of what constitutes a good matrix. A good matrix generally should have two functions-desorption and ionization of an analyte upon laser irradiation. One often tries to search for a chemical compound which provides both of these functions, but results show that such chemical compounds are very few. On the other hand, little attention has been given to the possibility of constructing a good matrix by blending two components, chosen so that one component serves for desorption and the other for protonation/deprotonation. Because of this lack of attention, many areas have been left unexplored. Cume and Yates first pursued the possibility of a two-component matrix using an ammonium salt. They concluded that the addition of salts, such as ammonium acetate, to a UV-absorbing matrix can have a positive effect on the sensitivity, reproducibility, and mass range in the MALDI analysis of oligodeoxynucleotides. They also pointed out that ammonium salts may serve to stabilize the phosphodiester backbone in deoxyoligonucleotides such that self-protonation can occur. However, the ...
The most common mutation of the cystic fibrosis gene is characterized by the deletion of three nucleotides that code phenylalanine in the 508 position of the cystic fibrosis transmembrane conductance regulator. We report the first measurements by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry for the delta F508 mutation in cystic fibrosis carriers and patients. Furthermore, in a blind test, results from the normal and delta F508 mutant alleles in 30 clinical samples based on MALDI mass spectrometry and on conventional gel analysis of the DNA were in total agreement. These results demonstrate the utility of MALDI mass spectrometry in the molecular diagnosis of mutant alleles and point to its potential use for ultra-fast detection in large-scale screening of DNA mutations.
Matrix-assisted laser desorption/ionization (MALDI) was used for several small proteins (such as insulin) and for peptides. It was found that the detection efficiencies of MALDI for the insulin B chain and the insulin A chain are drastically different. Similar phenomena were also observed for various types of peptides. The positive-ion signal of MALDI in detecting proteins or peptides was found to be greatly enhanced by the presence of a basic amino acid in their chains. The experimental results indicate that this enhancement may arise from proton transfer in solution by an acid-base reaction between the protein/peptide and matrix molecule. This pre-protonated mechanism provides a low energy barrier for the ionization of peptides in a MALDI process, and greatly reduces the energy threshold of MALDI. Matrix effects on the ionization mechanism are discussed.
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