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LC-MS/MS-Based, Label-Free, Peptide-Centric, Quantitative Proteomics of the H2O2 Stimulon in Wild-Type S. Cerevisiae Cells vs Those Deleted for the H2O2 Sensor, Ccp1

Title:

LC-MS/MS-Based, Label-Free, Peptide-Centric, Quantitative Proteomics of the H2O2 Stimulon in Wild-Type S. Cerevisiae Cells vs Those Deleted for the H2O2 Sensor, Ccp1

Sadeghian, Tanaz (2022) LC-MS/MS-Based, Label-Free, Peptide-Centric, Quantitative Proteomics of the H2O2 Stimulon in Wild-Type S. Cerevisiae Cells vs Those Deleted for the H2O2 Sensor, Ccp1. Masters thesis, Concordia University.

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Abstract

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) can be harmful to cells. However, cells adapt to sublethal doses of H2O2 by the enhancement of antioxidant synthesis and shifting their carbon flux to NADPH generation. Cytochrome c peroxidase (Ccp1) is a heme-based protein located in the intermembrane space of yeast mitochondria that functions as a sensor of H2O2. To fully explore the H2O2-sensing role of Ccp1, we compared the H2O2 stimulon in wt and ccp1Δ Saccharomyces cerevisiae cells (BY4741 strain) using label-free peptide-centric, quantitative proteomics. Cells were grown to mid-log phase, challenged with 0.4 mM H2O2, and whole-cell lysates were prepared 10 and 60 min later for three biological replicates. All the proteins in each lysate were concentrated into a single band on a 6% stacking gel for tryptic digestion and peptide analysis by nanoLC-MS/MS. A total of 2222 proteins were quantified and identified from their unique peptides (14,642) in the biological replicates. The stimulated and repressed proteins were analyzed using an abundance ratio (R) calculated by Proteome Discoverer v2.4, and Ccp1’s role was evaluated through the examination of proteins up- and downregulated following H2O2 challenge. Our results suggest that Ccp1 influences the abundance of key antioxidant enzymes such as Ctt1, Trx2 and Prx1 by effecting the levels of transcription factors involved in H2O2 signaling, including Yap1 and Hsf1. Ccp1 deletion alters the abundance of proteins that are important in NADPH production such as Rki1 and Sol3. These proteomics results combined with published biochemical data provide new insights into how Ccp1 contributes to the coordination of cellular defensive mechanisms and the shifts in metabolic pathways following H2O2 challenge.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (Masters)
Authors:Sadeghian, Tanaz
Institution:Concordia University
Degree Name:M. Sc.
Program:Chemistry
Date:July 2022
Thesis Supervisor(s):English, Ann
ID Code:990957
Deposited By: Tanaz Sadeghian
Deposited On:27 Oct 2022 14:24
Last Modified:29 Aug 2024 00:00

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