Date of Award
2018
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Dwight E. Matthews
Abstract
Heart failure is a major public health issue, with its prevalence estimated to be 6.5 million adults in the USA. Of the hospitalized heart failure (HF) cases, 50% are characterized by preserved ejection function (HFpEF). In HFpEF, the heart pumps a normal proportion of blood that enters it. However, thickening of the ventricular walls inhibits the chamber filling to normal volume. The direct basis of HFpEF is a slowed elongation of the cardiac muscle during the diastolic phase of the cardiac cycle. Elucidation of mechanisms that mediate relaxation of cardiac muscle could help understand the pathogenic mechanisms in HFpEF.
Myocardial contraction and relaxation are tightly controlled processes that involve thick and thin filament regulatory proteins. β-Adrenergic signaling pathway is a major regulator of myocardial contraction and relaxation via the activation of protein kinase A (PKA). Two key myofilament proteins, troponin I (TnI) and myosin binding protein-C (MyBPC), are phosphorylated by PKA following β-adrenergic stimulation. The purpose of this thesis is to develop a liquid chromatography-mass spectrometry (LC-MS) method for the quantification of phosphorylation in TnI and MyBPC and measure the changes in the degree of phosphorylation in transverse-aortic constriction (TAC) mouse hearts, a model representing HFpEF, and sham (control) mouse hearts.
The initial approach of the project was to develop a method for quantification of phosphopeptides using synthesized stable isotope labeled (SIL) peptides, both with and without phosphate modification. To accomplish this goal, a multiple reactions monitoring (MRM)-LC-MS method for the quantification of the synthesized SIL peptides was first developed. This method, using low picomole amounts, is applicable to researchers in the field using SIL peptides for quantification. However, when the SIL peptides were actually applied, we determined that there was a selective absorption of some phosphate peptides in the LC column, limiting the use of the SIL peptides for quantification. This result is also of general interest to others trying to identify phosphopeptides, not realizing that some peptides will go unmeasured. Thus, we returned to expanding an earlier method developed in our research group to quantify the degree of phosphorylation. Key to this work was the development of a quantification method directly from heart myofibrillar protein preparations without requiring isolation of individual proteins by gel electrophoresis.
Using the LC-MS method developed, we quantified phosphorylation sites of TnI and MyBPC in the TAC and control mouse hearts. The phosphorylation measurements showed no significant difference in phosphorylation between the TAC and control mice, except for one site, S302 in MyBPC that had a 13% decrease in phosphorylation with TAC. We conclude that in our TAC model, PKA dysfunction may not play a role in the initial development of HFpEF.
Language
en
Number of Pages
227 p.
Recommended Citation
Nukareddy, Praveena, "Quantification Of Mouse Cardiac Troponin I And Myosin Binding Protein C Phosphorylation By Liquid Chromatography-Mass Spectrometry (lc-Ms)" (2018). Graduate College Dissertations and Theses. 986.
https://scholarworks.uvm.edu/graddis/986