Abstract
To characterize protein phosphorylation in developing seed, a large-scale, mass spectrometry-based phosphoproteomic study was performed on whole seeds at five sequential stages of development in soybean (Glycine max), rapeseed (Brassica napus), and Arabidopsis (Arabidopsis thaliana). Phosphopeptides were enriched from 0.5 mg of total peptides using a combined strategy of immobilized metal affinity and metal oxide affinity chromatography. Enriched phosphopeptides were analyzed by Orbitrap tandem mass spectrometry and mass spectra mined against cognate genome or cDNA databases in both forward and randomized orientations, the latter to calculate false discovery rate. We identified a total of 2,001 phosphopeptides containing 1,026 unambiguous phosphorylation sites from 956 proteins, with an average false discovery rate of 0.78% for the entire study. The entire data set was uploaded into the Plant Protein Phosphorylation Database (www.p3db.org), including all meta-data and annotated spectra. The Plant Protein Phosphorylation Database is a portal for all plant phosphorylation data and allows for homology-based querying of experimentally determined phosphosites. Comparisons with other large-scale phosphoproteomic studies determined 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; other categories are only overrepresented in our study, like embryonic development. This investigation shows the importance of analyzing multiple plants and plant organs to comprehensively map the complete plant phosphoproteome.
Highlights
To characterize protein phosphorylation in developing seed, a large-scale, mass spectrometry-based phosphoproteomic study was performed on whole seeds at five sequential stages of development in soybean (Glycine max), rapeseed (Brassica napus), and Arabidopsis (Arabidopsis thaliana)
The coupling of phosphopeptide enrichment strategies to high-resolution mass spectrometry has led to a renaissance in phosphorylation site mapping, and several recent large-scale studies have expanded the knowledge of phosphorylation in plants (Sugiyama et al, 2008; Reiland et al, 2009; Grimsrud et al, 2010; Nakagami et al, 2010)
Phosphopeptide Identification from Developing Seeds. Phosphopeptide enrichment using both Fe-immobilized metal affinity chromatography (IMAC) and Ti-hydroxy acid-modified metal oxide chromatography in series was performed on three species, Arabidopsis, rapeseed, and soybean, at five stages of seed development in biological triplicate
Summary
To characterize protein phosphorylation in developing seed, a large-scale, mass spectrometry-based phosphoproteomic study was performed on whole seeds at five sequential stages of development in soybean (Glycine max), rapeseed (Brassica napus), and Arabidopsis (Arabidopsis thaliana). More targeted studies have identified several protein kinases involved in seed development. To better characterize the extent of protein phosphorylation during seed maturation, we performed a large-scale phosphorylation study on developing seeds from three species: Arabidopsis, rapeseed (Brassica napus), and soybean (Glycine max).
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