Cutting Edge Seminar - Jay J. Thelen: Phosphoproteomic analysis of seed maturation – from discovering phosphorylation sites to identifying kinase clients

UPSC Seminar Series 2014
Cutting Edge Seminar 

Speaker:
Jay J. Thelen
Department of Biochemistry, 
Christopher S. Bond Life Sciences Center
University of Missouri, Columbia, MO, USA

Title:
Phosphoproteomic analysis of seed maturation – from discovering phosphorylation sites to identifying kinase clients

Host: Vaughan Hurry

Place: Lilla hörsalen, KB3A9

Abstract

Although metabolic networks for storage reserve synthesis have been largely characterized in diverse plant seed we are only beginning to understand the complex regulatory processes involved in seed development. Protein phosphorylation is a major form of post-translational regulation in eukaryotes as evidenced by over 1000 protein kinases in the Arabidopsis proteome. To begin studying protein phosphorylation in developing seed we performed large-scale, mass spectrometry-based phosphoproteomic studies on seeds at five stages of development in soybean (Glycine max), rapeseed (Brassica napus), and Arabidopsis (Arabidopsis thaliana). Phosphopeptides were enriched from 0.5 mg total peptides using a combination of immobilized metal affinity and metal oxide affinity chromatography. Enriched phosphopeptides were analyzed by Orbitrap tandem mass spectrometry and spectra mined against cognate genome or cDNA databases in both forward and randomized orientations, the latter to calculate false discovery rate (FDR). We identified a total of 2001 phosphopeptides containing 1026 unambiguous phosphorylation sites from 956 proteins with an average FDR of 0.78% for the entire study (Meyer et al., 2012). The dataset was uploaded into the Plant Protein Phosphorylation Database (P3DB, www.p3db.org), including annotated spectra, for public accession. P3DB is a portal for all plant phosphorylation data and allows for homology-based querying of experimentally-determined phosphosites (Gao et al., 2009). Comparisons to other large-scale studies revealed that 652 of the phosphoproteins are novel to this study. The unique proteins fall into several Gene Ontology categories, some of which are overrepresented in our study as well as other large scale phosphoproteomic studies including metabolic process and RNA binding; while other categories are only overrepresented in our study like embryonic development. Leveraging large-scale phosphoproteomic datasets such as these, we developed a phosphorylation prediction tool called MUSite (http://musite.sourceforge.net/) that incorporates protein disorder as one of three features for prediction (Gao et al., 2010). The sensitivity and accuracy of this prediction algorithm is unmatched, and application to whole plant proteomes such as Arabidopsis TAIR10 indicates greater than 17,000 phosphorylation sites at the 99% confidence interval. Clearly, experimental and bioinformatic prediction of phosphorylation sites is rapidly becoming a facile task. However, confirmation and identification of cognate protein kinases responsible for these events remains challenging. I will also introduce a novel approach to address this problem called the Kinase Client or KiC Assay (Huang et al., 2009). Using this approach we have identified many kinase client relationships, including three different protein kinases responsible for over ten phosphorylation events on a single phosphoprotein.

Ahsan N, Huang Y, Tovar-Mendez A, Swatek KN, Zhang J, Miernyk JA, Xu D, Thelen JJ. (2013) A

Versatile Mass Spectrometry-Based Method to Both Identify Kinase Client-Relationships and Characterize Signaling Network Topology. J Proteome Res. 12:937-48

Meyer LJ, Gao J, Xu D, Thelen JJ (2012) Phosphoproteomic analysis of seed maturation in

Arabidopsis, rapeseed, and soybean. Plant Physiol. 159:517-28

Huang Y, Houston NL, Tovar-Mendez A, Stevenson SE, Miernyk JA, Randall DD, Thelen JJ (2010) A

quantitative mass spectrometry-based approach for identifying protein kinase –clients and quantifying kinase activity. Anal. Biochem. 402:69-76

Gao J, Agrawal GK, Thelen JJ, Xu D (2009) P3DB: A plant protein phosphorylation database. Nucl. Acids

Res. 37:D960-962

Gao J, Thelen JJ, Dunker AK, Xu D (2010) Musite: a tool for global prediction of general and kinase-

specific phosphorylation sites. Mol. Cell. Prot. 9:2586-25600