The use of active substrates and membranes in matrix-assisted laser desorption/ionization mass spectrometry.

This work has been focused on (1) the development and use of active substrates as on-probe tip purification devices for MALDI/MS analysis of DNA and proteins, (2) development of methods using membranes for the characterization of proteins, and (3) characterization of new MALDI/MS matrices. Chapter 3 reports two new MALDI matrices, indole-3-pyruvic acid and 3-hydroxy-4-methoxyphenylpyruvic acid, for protein/peptide detection. Several factors affecting the effectiveness of a MALDI matrix are discussed. In particular, photon/thermal stability of a matrix material may be a critical factor. The idea and use of active substrates as on-probe tip purification devices is introduced in Chapter 4. Two substrates based on Nafion$\sp{\rm TM}$ and nitrocellulose were identified and used for effective detection of DNA in biological samples and proteins in crude biological fluids such as in milk and bacterial cell lysates. The mechanisms of using such active substrates were identified mainly as ion exchange and hydrophobic interactions. Technical aspects involved in the use of active substrates are discussed. MALDI/MS detection of both ss-DNA (up to 73-mer) and ds-DNA (9 to 622-bp) is presented in Chapter 5. Difficulties in MALDI/MS detection of DNAs is primarily due to the presence of cationic impurities in typical biological samples. Active substrates were used for the detection of DNA fragments (up to $\sim$600 bases) obtained from PCR reactions and enzymatic digests. Chapter 6 establishes a protocol for the detection of DNA polymorphisms based on MALDI/MS on active substrates using model genes including carbonic anhydrase and cystic fibrosis transmembrane conductance regulator (CFTR) genes. Chapter 7 explores the use of a nitrocellulose membrane as an interface between MALDI/MS and SDS-PAGE because of its excellent retention property for proteins and peptides. Proteins transferred to nitrocellulose from an SDS-PAGE gel can be efficiently detected with MALDI/MS by directly dissolving the membrane in matrix solutions prepared in organic solvents to generate molecular weight and peptide map information. This procedure offers improvement in sensitivity over conventional dye staining methods.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105169/1/9635646.pdfDescription of 9635646.pdf : Restricted to UM users only