Type of Document	Dissertation
Author	Al-Sayah, Mohammad Ahmed
URN	etd-11102004-162015
Title	New Strategies for Proteomics and Peptidomics using Polymer Liquid Crystals for Electophoresis
Degree	Doctor of Philosophy
Department	Chemistry and Biochemistry, Department of
Advisory Committee	Advisor Name Title Randolph Rill Committee Chair David Van Winkle Committee Member John G. Dorsey Committee Member Joseph B. Schlenoff Committee Member
Keywords	Cascade Yellow Peptidomics Proteomics Gel Electrophoresis Pluronic F127 Mass Spectrometry
Date of Defense	2004-10-27
Availability	unrestricted
Abstract Different gel matrices were explored to extend the use of two dimensional (2D) gel electrophoresis for peptide analysis. Excellent separations of peptides labeled with the fluorescent dye Cascade Yellow were achieved in one dimension on two gel media: traditional polyacrylamide and reversible liquid crystalline gels of Pluronic F127. Separations on both media depended primarily on size to charge ratio, excepting a few peptides strongly retained by Pluronic F127. A unique 2D gel electrophoresis system for peptide separation coupled with MALDI-TOF mass spectrometry for identification was developed. Cascade Yellow succinimidyl ester, an amine-reactive dye, labels the amino-terminus of peptides and the å-amino group of lysines at pH 7-9. Occasional labeling of tyrosine residues was also observed. Specific amino-terminal labeling was achieved at alkaline pH (pH >10) due to the base-lability of the å-amino and the tyrosine adducts. The 2D system utilized 15% polyacrylamide with the basic (pH 8.3) Laemmli buffer system (without SDS) in the first dimension. Pluronic F127 (24%) was used in the second dimension with acidic Tris-ClCH2COOH buffer (pH 3.0). The second dimension in Pluronic F127 was done horizontally with a thin overlayer of buffer to provide direct access to the separated peptides. Due to its semi-fluid nature, Pluronic F127 provided a good interface between the two dimensions so that the peptides migrated smoothly from the first dimension to the second. The peak capacity of the 2D mini-gel system (8x10 cm) was approximately 500. Larger gels are expected to yield a peak capacity of about 2000, competitive with many 2D HPLC methods. MALDI-TOF MS was used to identify peptides in spots directly sipped from gels. Peptide samples with concentrations > 0.5 ìg/ml were directly spotted on MALDI targets and identified without further purification. Small polymer chains contaminating Pluronic F127 started to interfere with the detection of peptides at concentrations < 0.5 ìg/ml. Sensitivity of MALDI-TOF MS identification of peptides was increased about 10-fold when Pluronic F127 used for electrophoresis was purified by gel permeation chromatography to remove short polymer chains. This 2D system is applicable to robotic sampling and should facilitate protein identification and peptidomics.
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