Abstract

Characterization of phosphoproteins-including detection, identification of phosphoproteins and identification of phosphorylation sites-is mostly done with radiolabeling and proteomic techniques. Three main topics related to phosphoprotein characterization are included in this dissertation. First, large-scale characterization of the CHO (Chinese hamster ovary) cell phosphoproteome was done using two dimensional gel electrophoresis (2DE) separation, visualization of phosphoproteins by radiolabeling or a phosphoprotein specific dye, followed by MALDI-TOF identification. Because radiolabeling of phosphoproteins is very sensitive and straightforward to quantify, such analysis can give a clear picture of the relative phosphosphorylation of proteins present in a sample. But there are also limitations to this approach, such as the inability of 2DE to separate hydrophobic, acidic and large proteins and the poor detection limits of common protein stains such as Coomassie stain. Additionally, it is difficulty to excise the right spots for identification because of the low abundance of phosphoproteins which have been visualized by radiolabeling. Furthermore, there are problems associated with metabolic radiolabeling. A second topic of the dissertation is the development of a novel strong cation exchange monolithic column for MudPIT (multidimensional protein identification technology) and phosphopeptide isolation. This column, a poly(AMPS-co-PEGDA) monolith containing as high as 40% AMPS, has several favorable features, such as high binding capacity, extraordinarily high resolution, and high peak capacity, making it ideal for resolving complex peptide samples. Application of this novel column to isolate model phosphopeptides was shown. More general use of this column in MudPIT (strong cation exchange column followed by reverse-phased MS/MS) is probably somewhat limited, due to the hydrophobicity of the AMPS monomer. A better monolith could be obtained if a more hydrophilic monomer was used. In the third area of the dissertation, several individual protein phosphorylation sites were analyzed, employing different strategies. Phosphorylation sites of one multiply phosphorylated tryptic peptide from CK2-phosphorylated phosducin-like protein (PhLP) was well characterized using enrichment with a MonoTip® TiO Pipette Tip. Analysis of syntaxin 1a phosphorylation by AMPK (AMP-activated protein kinase) was done by peptide level mapping for potential phosphopeptides after its unsuccessful trial with enrichment using the MonoTip® TiO Pipette Tip. Several criteria such as existence of non-phosphorylated forms of potential phosphopeptides, controls and reasonable retention times were used to rule out false positives. Phosphorylation of syntaxin 1a by AMPK was narrowed down to tryptic peptide T32 with evidence from different sources. Three phosphorylation sites of syntaxin 4 by AMPK were identified within the same peptide (Q65QVTILATPLPEESMK80). Further pinpointing of phosphorylation site(s) for syntaxin 1a by AMPK and further confirmation of these phosphorylation sites in syntaxin 4 by AMPK are required in vivo. The role of phosphorylation in syntaxin 4 by AMPK is the next step toward elucidation of AMPK activation and regulation of the glucose uptake mechanism.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

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