UVM Theses and Dissertations
Format:
Print
Author:
Previs, Michael J.
Title:
Dept./Program:
Cell and Molecular Biology Program
Year:
2010
Degree:
Ph. D.
Abstract:
Heart failure is a symptomatic syndrome that is one of the greatest public health problems in the United States. Heart failure negatively affects daily living by limiting a person's ability to perform physical activity and ultimately could lead to death. The progression of the symptoms of heart failure is highly correlated to a remodeling process within the heart and limited contractile function. This cardiac remodeling process involves changes in whole heart morphology as well as molecular alterations of both the contractile and calcium handling proteins. One of many potential alterations that occur during heart failure includes the phosphorylation of a thin filament protein or proteins. The thin filament proteins are directly involved in regulating the response of the contractile apparatus to calcium ions, and thus they are intimately involved with the regulation of heart contraction.
Protein phosphorylation is known to modulate protein function in many biological systems. However, the precise mechanisms by which phosphorylation alter function are difficult to study due to the complexity of multiple phosphorylation events and lack of simple molecular assays to identify and quantify protein phosphorylation. Therefore, the first specific aim of this thesis is to develop a mass spectrometry based method for the identification and quantification of peptide specific protein phosphorylation and demonstrate the accuracy and precision of this technique using samples of known degrees of phosphorylation. The second specific aim of this thesis is to directly identify the protein or proteins that are phosphorylated in muscle biopsies obtained from failing and non-failing human hearts and quantify the degree of phosphorylation.
The liquid chromatography mass spectrometry based methodology described in this thesis provides critical insight into the sample workflow and has potential applications beyond the study of protein phosphorylation. The application of this analytical technique to study phosphorylation ofthin filaments from the human heart was shown to be successful in the quantification of changes in the phosphorylation ofmultiple sites in troponin 1. We found that troponin I Ser-23 and Ser-24 were phosphorylated in failing and non-failing hearts, and the degree of phosphorylation was greater in nonfailing hearts compared to end-stage failing hearts (64±3.6% and 31±6.2%, p <0.005). Although there was no significant difference between groups, troponin I from 3 of the 6 failing hearts were assumed to contain phosphorylation on Ser-42 and/or Ser-44 (54±7.5% and 16±4.4%, p <0.01).
It was also discovered that Ser-283, a novel site in the thin filament regulatory protein a-tropomyosin, is phosphorylated in the human heart. The degree of phosphorylation was significantly greater in the failing hearts (45±2.6% and 26±3.9%, p <0.005). These findings provide mechanistic insight into the role of phosphorylation in the modulation of cardiac function and exciting avenues for future research. In summary, this thesis highlights the unique ability to use quantitative mass spectrometry to study phosphorylation events that alter contractile function. This mass spectrometry based approach to identify and quantify molecular alterations in the human heart may provide direct targets for therapeutic reagents to treat heart failure and enhance people's lives.
Protein phosphorylation is known to modulate protein function in many biological systems. However, the precise mechanisms by which phosphorylation alter function are difficult to study due to the complexity of multiple phosphorylation events and lack of simple molecular assays to identify and quantify protein phosphorylation. Therefore, the first specific aim of this thesis is to develop a mass spectrometry based method for the identification and quantification of peptide specific protein phosphorylation and demonstrate the accuracy and precision of this technique using samples of known degrees of phosphorylation. The second specific aim of this thesis is to directly identify the protein or proteins that are phosphorylated in muscle biopsies obtained from failing and non-failing human hearts and quantify the degree of phosphorylation.
The liquid chromatography mass spectrometry based methodology described in this thesis provides critical insight into the sample workflow and has potential applications beyond the study of protein phosphorylation. The application of this analytical technique to study phosphorylation ofthin filaments from the human heart was shown to be successful in the quantification of changes in the phosphorylation ofmultiple sites in troponin 1. We found that troponin I Ser-23 and Ser-24 were phosphorylated in failing and non-failing hearts, and the degree of phosphorylation was greater in nonfailing hearts compared to end-stage failing hearts (64±3.6% and 31±6.2%, p <0.005). Although there was no significant difference between groups, troponin I from 3 of the 6 failing hearts were assumed to contain phosphorylation on Ser-42 and/or Ser-44 (54±7.5% and 16±4.4%, p <0.01).
It was also discovered that Ser-283, a novel site in the thin filament regulatory protein a-tropomyosin, is phosphorylated in the human heart. The degree of phosphorylation was significantly greater in the failing hearts (45±2.6% and 26±3.9%, p <0.005). These findings provide mechanistic insight into the role of phosphorylation in the modulation of cardiac function and exciting avenues for future research. In summary, this thesis highlights the unique ability to use quantitative mass spectrometry to study phosphorylation events that alter contractile function. This mass spectrometry based approach to identify and quantify molecular alterations in the human heart may provide direct targets for therapeutic reagents to treat heart failure and enhance people's lives.