Characterization of protein phosphorylation by mass spectrometry

Phosphorylation is a post-translational modification that dynamically controls protein-protein interactions, transduces cellular signaling cascades, and regulates gene expression. Studying protein phosphorylation using molecular biology methods, such as Western blots or Phos-tag gel electrophoresis, require highly specific primary antibodies or purified samples. Neither of these methods can confirm the primary sequence of the labeled protein nor can they distinguish phosphorylation sites. Mass spectrometry is the gold standard analytical technique used to determine protein amino acid sequence and phosphorylation state. Proteins can be analyzed by mass spectrometry intact or as digested peptides. Both approaches have merits for studying phosphorylation. In Chapter 2, intact protein analyses were employed for the investigation of histone H1 phosphorylation in human neuronal cells. This approach enabled the identification of progressive phosphorylation sites on three distinct H1 subtypes. In Chapter 3, phosphorylation of the plant protein RGA was investigated using peptide-based standard proteomics analyses, which enabled the detection of multiple low-abundance post-translational modifications. These experiments identified that RGA is phosphorylated by the kinase CDK8, which was not previously known. In Chapter 4, a new method was developed to stabilize calcium-modified peptides during electrospray ionization. These analyses determined that calcium-modification is pH sensitive, and that charge enhancement by calcium improves phosphorylation site-localization on charge-sparse peptides.