Submitted to the Department of Chemical and Systems Biology.
Thesis (Ph.D.)--Stanford University, 2014.
Global analysis of post-translational modifications (PTMs) by mass spectrometry facilitates interrogation of complex signaling networks. We have utilized this technology and developed analytical tools to combine peptide level data into high confidence PTM site identifications and to assess the quality of quantification measurements. We have applied this workflow in order to study three systems: 1) mitochondrial acetylation and phosphorylation during cardiac ischemia, 2) phosphorylation regulation Toxoplasma gondii egress from host cells, and 3) early phosphorylation events during hedgehog signaling. 1. Acetylation is a highly abundant modification in the mitochondrion, but we lack a general understanding of both its regulation and functional relevance, especially with regard to the acetyltransfer mechanism. To explore the nature of mitochondrial acetylation, we compared several features of mitochondrial phosphorylation and acetylation sites, showcasing stark differences between these two PTMs. We found that mitochondrial acetylation is biased towards ordered secondary protein structures, and that acetylation and succinylation modify the same residues in an unbiased, unenriched sample. We suggest that these results are consistent with low levels of background, non-functional acetylation. In order to understand acetylation regulation, we must know the mechanism of acetyl transfer. Our findings indicate that either a putative mitochondrial acetyltransferase or family of acyltransferases exist that operate non-specifically and recognize substrates in a matter entirely distinct from kinases, or else that a substantial fraction of observed mitochondrial lysine acylation results from non-enzymatic modification by acyl-CoAs. 2. We report the first phosphoproteomes of T. gondii and P. falciparum. These datasets demonstrated for the first time tyrosine phosphorylation in P. falciparum, and unusual phosphorylation-site motifs for P. falciparum. We observed extensive phosphorylation beyond the intercellular parasites' boundaries. In a follow-up study in T. gondii, we focused on the role of calcium-dependent protein kinase 3 (TgCDPK3), which regulates parasite egress from the host cell in the presence of a calciumionophore. We measured relative phosphorylation site abundance in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling by amino acids in cell culture (SILAC). Our results demonstrate that TgCDPK3 regulates several functions in addition to egress. We also observed novel phosphorylation events on proteins predicted to play a role in egress, and which implicate TgCDPK3 in regulating other calcium-dependent signaling pathways, including TgCDPK1. 3. Finally, we have initiated a study of the early phosphorylation signaling events in response to hedgehog signaling. Here, we report preliminary results that implicate a kinase, casein kinase 2 (CK2), in hedgehog signaling. Work is currently being undertaken in the Scott Lab to further characterize the role of CK2 in hedgehog signaling while we analyze biological replicates to expand proteome coverage and validate our initial hits.