Fundamental studies of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and its applications to the analysis of biological materials.

Abstract

This work is focused on the fundamental studies of matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and its application in the detection of biological materials including oligonucleotides, proteins and peptides. The fundamental studies include: the use of active substrates for the detection of DNA in which a modified Nafion film substrate was used to improve the detection of DNA, especially for DNA strands greater than 200 base pairs; the characterization and evaluation of two new matrices (4-hydroxy-3-methoxyphenylpyruvic acid HMPA and indole-3-pyruvic acid EPA) for MALDI/MS in the detection of proteins and peptides; and improvement of mass resolution of MALDI TOF MS by using a simple high voltage transistor switch which can provide high mass resolution up to 1000 for oligonucleotides up to a 60-mer; and by the modification of the oligonucleotide backbone via alkylation of the -S<super>-</super> group on the phosphorothioate oligonucleotides to increase the stability of the oligonucleotide against fragmentation. We also investigated applications of MALDI/MS for the detection of DNA, proteins and peptides which include: development of methodologies for rapid screening of genetic polymorphisms using buccal. cell DNA with detection by MALDI/MS where MALDI/MS can provide more accurate and rapid analysis than the traditional gel electrophoresis method; and development of procedures for off-line coupling between liquid separation methods (CZE and CIEF) with MALDI TOF MS and its applications to the detection of protein and peptide mixtures.