Novel applications of bioanalytical techniques for proteomic research

Shelly Jo Dormady, Purdue University

Abstract

Following a brief presentation of a popular methodology currently employed to diagnose diseases, a specific technique, Enzyme-Linked Immunosorbent Assay (ELISA), which employs this methodology is introduced. The advantages of using a waterfall enzyme cascade for ELISA detection over the commonly employed single enzymatic detection system were explored. The results indicate that only slight improvements in detection can be achieved with this implementation. An alternative methodology to diagnosis is then presented. A newly emerging field of research which employs this methodology is Proteomics. It is envisioned that with Proteomics, the entire protein complement of organism's genomes can be identified. At the forefront of Proteomic efforts a reduction in the massive number of proteins will be necessary. This reduction was explored through the selection of peptide fragments which contain the rare amino acid cysteine. Positive confirmation of this selection was achieved with the peptide fragments of a single protein. As equally important in Proteomic efforts will be the use of mass spectrometry. In particular, the mass spectral analysis of complex peptide mixtures. The role of spectral suppression due to both varying concentration and amino acid composition of the peptide fragments were explored. The results indicate that both of the above mentioned factors are equally important to consider. Finally, the advantage of using an on-column, in contrast to a free solution, enzymatic digest to generate peptide fragments was studied. The results indicate that in addition to enhanced proteolysis, peptide fragments generated via an on-column process are free of disulfide bond scrambled artifacts.

Degree

Ph.D.

Advisors

Regnier, Purdue University.

Subject Area

Analytical chemistry|Biochemistry

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