Characterization of Post-Translationally Modified Peptides and Proteins Using Lanthanide-Based Labeling Strategies

• Main Author: Gant-Branum, Randi Lee
• Other Authors: David E. Cliffel
• Format: Others
• Language: en
• Published: VANDERBILT 2011
• Subjects: Chemistry
• Online Access: http://etd.library.vanderbilt.edu/available/etd-07202011-193844/

Characterization of Post-Translationally Modified Peptides and Proteins Using Lanthanide-Based Labeling Strategies By Randi Gant-Branum Abstract: Numerous biological processes are regulated by the stoichiometry of protein phosphorylation. Current mass spectrometry (MS) based strategies for quantifying sites of protein phosphorylation include stable isotope techniques that take advantage of mass shifts provided by isotopologues. Lanthanide-based labeling strategies allow for greater mass separation than current isotope-based strategies due to incorporation of lanthanide metals of greater mass differences (2-36 Da), but have not been previously demonstrated for selective phosphopeptide and protein quantitation. Furthermore, lanthanide-based labelling strategies may be used as mobility shift anchors for rapid visualization in ion mobility-mass spectrometry (IM-MS) analysis. In this dissertation, I demonstrate how lanthanide-based labeling of phosphorylated peptides and proteins facilitates rapid identification, relative quantitation, and phosphorylation site identification of phosphorylated peptides and proteins in complex mixtures. In this labeling, phosphorylated peptides are selectively modified at the phosphorylation site via beta-elimination/anionic thiol Michael addition chemistry. In this manner, phosphorylated peptides are converted to cysteine-like residues, which then readily react with cysteine-specific labels. A lanthanide-chelating label is added via maleimide chemistry and selected lanthanide metals are subsequently chelated to a macrocycle moiety. Phosphorylated peptides are then visually identified, quantitated, and fragmented to provide comprehensive analysis. To demonstrate this technique, phosphorylated peptides and protein mixtures from proteolytic digestion are identified and quantitated in various molar ratios with comparable sensitivity and relative error (~10%) to current isotopologue-based relative quantitation strategies. Moreover, profiling relative stoichiometry between reciprocal glycosylation and phosphorylation is also desired when these modifications share the same sequence position. This labeling technique was applied to O-linked glycosylated peptides in benchmarking experiments to evaluate its labeling efficiency. Interesting future applications of this labeling strategy are also proposed.