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Title:Applications of mass spectrometry to protein characterization
Author(s):Huang, Rong
Doctoral Committee Chair(s):Rinehart, Kenneth L., Jr.
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Chemistry, Analytical
Chemistry, Biochemistry
Abstract:The application of mass spectrometry as a protein sequencing method is demonstrated by the study of bovine kidney aldose reductase. The protein is blocked at the N-terminus and is not amenable to Edman degradation. Using a strategy of homology comparison, the amino acid sequence was determined solely by mass spectrometric methods. The nature of aldose reductase oxidation was also identified.
In combination with DNA cloning and sequencing techniques, mass spectrometric methods were designed to verify and correct DNA-derived amino acid sequences and provide post-translational modification information. The study involved cytochrome P450 proteins, including P450$\rm\sb{lin}$ and P450$\rm\sb{cam}$ from Pseudomonas putida. The results indicated modifications that were not observed by x-ray crystallography. Although surprising, it serves as a good example demonstrating the advantages of different techniques.
With recent advances in coupling HPLC with mass spectrometry, on-line LC/MS was applied to the analysis of proteolytic peptides from a variety of proteins, including bovine kidney aldose reductase, cytochrome P450$\rm\sb{lin},$ insect hemoglobin, and nitric oxide synthase. Peptide mapping of nitric oxide synthase provided information about the two functional domains of the protein, i.e., the oxygenase domain and the reductase domain. Attempts were also made to map the heme binding site of the oxygenase domain.
On-line LC/MS was extended to protein mixture analysis. The proteins involved in the study were hemoglobin components from Buenoa margaritacea. These proteins are structurally related and difficult to separate. The additional molecular weight information obtained from LC/MS was proven most valuable for the identification. On-line LC/MS also allowed investigation of the effects of environmental factors such as temperature on the hemoglobin compositions.
Besides covalent structure information, the potential of studying non-covalent structure using mass spectrometry was explored. Non-covalent protein structure was studied for a protein-heme complex. Factors affecting the mass spectrometric determination of non-covalent structures and the possible artifacts of the analysis were examined.
Issue Date:1995
Type:Text
Language:English
URI:http://hdl.handle.net/2142/20686
Rights Information:Copyright 1995 Huang, Rong
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9624370
OCLC Identifier:(UMI)AAI9624370


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