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 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 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 / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial
fulfillment of the requirements for the degree of Doctor of Philosophy. / Degree Awarded: Fall semester, 2004. / Date of Defense: October 27, 2004. / Cascade yellow, peptidomics, proteomics, gel electrophoresis, pluronic F127, mass spectrometry / Includes bibliographical references. / Randolph L. Rill, Professor Directing Dissertation; David H. Van Winkle, Outside Committee Member; John G. Dorsey, Committee Member; Joseph B. Schlenoff, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_168067 |
| Contributors | Al-Sayah, Mohammad Ahmed (authoraut), Rill, Randolph L. (professor directing dissertation), Winkle, David H. Van (outside committee member), Dorsey, John G. (committee member), Schlenoff, Joseph B. (committee member), Department of Chemistry and Biochemistry (degree granting department), Florida State University (degree granting institution) |
| Publisher | Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource, computer, application/pdf |
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