|Huang, Yadong -|
|Houston, Norma -|
|Tovar-Mendez, Alejandro -|
|Stevenson, Severin -|
|Randall, Douglas -|
|Thelen, Jay -|
Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 22, 2010
Publication Date: March 25, 2010
Citation: Huang, Y., Houston, N.L., Tovar-Mendez, A., Stevenson, S.E., Miernyk, J.A., Randall, D.D., Thelen, J.J. 2010. A Quantitative Mass Spectrometry-based Approach for Identifying Protein Kinase-Clients and Quantifying Kinase Activity. Analytical Biochemistry. 402(2010):69-76. Interpretive Summary: An important part of how plants respond to changes in the environment involves chemical modification of cellular proteins. In a living cell, thousands of different proteins can be chemically modified. Unfortunately, identifying which proteins are modified is expensive, time-consuming, and technically difficult. We have developed a relatively simple method to determine in the laboratory which proteins are chemically modified in living cells and where the modifications are located. The method was tested with a single protein and in mixtures of proteins, and it was shown to be very accurate. Results obtained using this method will allow us to better understand how plants respond to environmental changes. These results will be useful to other researchers in their efforts to improve agricultural production through both classical breeding and application of biotechnology-based strategies.
Technical Abstract: The Homo sapiens and Arabidopsis thaliana genomes are believed to encode >500 and >1,000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Mass spectrometry (MS)-based approaches have been integral to the large-scale mapping of protein phosphorylation sites. Herein we describe a quantitative MS-based approach that can be used for identifying kinase-client proteins and mapping phosphorylation sites. The method also has obvious utility for characterization of individual protein kinases. During method development we have used the dedicated kinase, pyruvate dehydrogenase kinase (PDK) for the invitro assays. Through the use of soluble peptide cocktails, we demonstrated that the assay is both sensitive and highly specific. Applying a label-free spectral counting-method, the activity of PDK was determined using the peptide substrate YHGH292SMSDPGSTYR, derived from the pyruvate dehydrogenase E1a subunit sequence. The utility of spectral counting was further validated by studying the negative effect of Met oxidation on peptide phosphorylation. We additionally measured the activity of the unrelated calcium-dependent protein kinase 3 (CPK3), demonstrating the utility of the method in protein kinase screening applications.