Characterization of Protein Sequence and Post-translational Modifications Using Mass Spectrometry 947 views

Proteins are large biological molecules consisting of one or more polypeptide chains with various amino acid sequences. The side chains of amino acids in a protein can be covalently modified by functional biochemical groups, a process known as post-translational modification (PTM). The structure and properties of a protein are essentially determined by the amino acid sequences and their PTMs. Mass spectrometry (MS) has emerged as the premier method for protein sequencing and PTM identification owing to its high sensitivity, accuracy, high analysis speed, and minimal bias. This dissertation describes the implementation of high performance liquid chromatography (HPLC) coupled with tandem MS using electron transfer dissociation (ETD) and collision-activated dissociation (CAD) as fragmentation methods to characterize sequence and PTMs of several proteins. Chapter 1 is an overview of the application of mass spectrometry to protein characterization. This is followed by Chapter 2, which reports a comprehensive mapping of phosphorylation on cytoplasmic dynein from rat neurons that is mainly involved in retrograde axonal transport such as neurotrophin signaling. This study revealed 35 phosphorylation sites on different subunits, representing the largest number of phosphorylation sites reported on cytoplasmic dynein in any single study. Specially, the identification of two neurotrophin-sensitive dynein phosphorylation sites led to the following studies to identify a mechanism that is used to recruit the cytoplasmic dynein to the signaling endosome upon neurotrophin stimulation for retrograde transport. Chapter 3 reports the identification of phosphorylation and methylation on truncated desmoplakin (DP) C-terminal tail. DP is a cytoskeletal linker protein that tethers the intermediate filament (IF) network to desmosome in the heart and skin, and is required for maintaining mechanical integrity of these tissues. The subsequent studies demonstrated that the crosstalk between arginine methylation and serine phosphorylation cascades tunes DP-IF interactions and promote desmosome assembly and cell adhesion. The last chapter reports “middle-down” MS characterization of a monoclonal antibody (mAb). This chapter involves the development of a novel “size-controlled” protein digestion enzyme reactor, which mainly generates 3-8 kDa large peptides from a protein. These large peptides yield high protein sequence coverage using ETD, facilitating the identification of sequences and PTMs of mAb.

PHD (Doctor of Philosophy)

mass spectrometry; protein

English

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2014-04-28