Matrix-assisted laser desorption/ionization mass spectrometric study of biological molecules.
Liu, Yan-Hui
1996
Abstract
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been widely applied in the study of large biomolecules due to its capability for producing large intact molecular ions of biomolecules, its high sensitivity, and its ability to analyze mixtures in the presence of biological impurities. In this thesis, a linear time-of-flight (TOF) mass spectrometer with an extended mass range and high ion transmission efficiency, was employed for the separation and detection of large biomolecular ions produced by the MALDI technique. Using this methodology, DNA samples up to over 600 bp could be detected and mass analyzed. The speed and accuracy of MALDI TOF MS make it a potential alternative to gel electrophoresis for the analysis of biomolecules, such as DNA and proteins. MALDI using an ion trap/reflectron time-of-flight mass spectrometer is also explored as a means for gas phase sequencing of oligonucleotides, where the backbone cleavage and the cleavage of the glycosidic bonds are routinely observed. In this work, two critical aspects in MALDI TOF MS analysis of biomolecules including low detection sensitivity and the interference of impurities from real biological samples during the MALDI process are addressed. To reduce the discrimination of the MCP detector toward heavy ions and fragmentation of the biomolecules during the acceleration and separation process, a Cu-Be postacceleration detector stage is used. This postacceleration scheme is found to be essential for the sensitive detection of DNA molecules $>$70 bp, which cannot otherwise be detected without it. An on-probe purification scheme involving the introduction of an active polymeric film/membrane substrate into the sample preparation is applied in MALDI experiments of DNA and proteins. Electrostatic interactions between the active surface of nitrocellulose (NC) or Nafion film and the impurities (e.g., cationic species and buffer) effectively exclude/reduce the interference of impurities during MALDI of DNA, which allows the routine application of MALDI TOF MS to large DNA fragments $>$600 bp. This sample preparation method was used in diagnostic analysis in terms of DNA mapping and screening for human disease genes such as cystic fibrosis, Tay-Sachs, and carbonic anhydrase at the femtomole detection level. The application of NC or PVDF membranes in protein/peptide analysis, based on protein, peptide-membrane hydrophobic interactions, also allows the MALDI TOF MS analysis of SDS-PAGE separated protein/peptides following elution or dissolution of the protein/peptides from the membrane. We could use this method to detect proteins from real cellular samples at the low picomole level that have been separated and isolated by SDS-PAGE.Other Identifiers
(UMI)AAI9635556
Subjects
Chemistry, Analytical Chemistry, Biochemistry
Types
Thesis
Metadata
Show full item recordCollections
Remediation of Harmful Language
The University of Michigan Library aims to describe library materials in a way that respects the people and communities who create, use, and are represented in our collections. Report harmful or offensive language in catalog records, finding aids, or elsewhere in our collections anonymously through our metadata feedback form. More information at Remediation of Harmful Language.
Accessibility
If you are unable to use this file in its current format, please select the Contact Us link and we can modify it to make it more accessible to you.