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Technology Project 1

Akhiesh Pandey and Robert Cotter


Phosphorylation and kinase tool development

Kinase/phosphatase signaling cascades are dysregulated in a large number of heart diseases, including HF and result in changes to the phospho-proteome of the myocyte. There is therefore a need to develop technology that will improve both the efficiency of identifying and quantifying phosphorylated proteins at the modified amino acid level. Equally important is determining the activity level of the kinases and phosphatases involved.  Many signaling cascades in the heart have yet to be identified or have been only poorly characterized in cardiac muscle.  This is especially important as most kinases and phosphatases are also modified by PTMs, including phosphorylation, O-GlcNAc and oxidation.

Overall Research Goal: To adapt existing methods and develop new tools for investigating phospho-regulation within the myocyte. Four different areas will be emphasized i) extensive and efficient characterization of the phospho-proteome, ii) quantification of the phosphorylation at each site, iii) identification of the kinases involved and iv) their corresponding activity.

Goal 1: Identification and quantification of phospho-proteome of cardiac myocytes including the myofilament and mitochondria subproteome.

Rationale: The fundamental importance of phosphorylation in regulation of the myocyte is well established and extensive phosphorylation of the mitochondrial subproteome is also beginning to be understood. However, there is a great need for a systematic and in-depth analysis to identify the phospho-proteome of the myocytes at the amino acid residue level. This information is required before functional assessment can begin.

Goal 2: Identification, quantification and activity of the kinase-specific phospho-proteome using peptide microarrays.

Rationale: Profiling of cardiac myocytes or tissue specifically for their kinase-driven pathways will be done using the novel approach of peptide arrays.

Goal 3: MRM quantification of known phosphorylation sites in human HF.

Rationale: MS based methods are desperately needed to allow for the easy and accurate quantification of different amino acid residues in the same or different proteins. Abs are rarely available for a specific phosphorylation sites. Hence, a proteomic method is needed that can allow easy quantification of ALL phosphorylation sites on a protein and is transferable to the broader community. MRM is a MS method that can obtain absolute concentration of multiple specific proteins and phospho-proteins in a complex mixture without the need for Abs. It is truly breakthrough technology. MRM is based on detection and quantification of signature peptides unique to a protein sequence). The advantages of MRM over quantitative western blots or ELISAs include: Ab free (unless enrichment is required), faster development, faster assays, limited fractionation, sensitivity. Multiplexing (including multiple phosphorylation sites) and precise absolute quantification. Absolute quantification is carried out by the addition of stable isotopically labeled peptides.