title: Sample preparation for phosphoproteomic analysis of circadian time series in Arabidopsis thaliana.
abstract: Systems biological approaches to study the Arabidopsis thaliana circadian clock have mainly focused on transcriptomics while little is known about the proteome, and even less about posttranslational modifications. Evidence has emerged that posttranslational protein modifications, in particular phosphorylation, play an important role for the clock and its output. Phosphoproteomics is the method of choice for a large-scale approach to gain more knowledge about rhythmic protein phosphorylation. Recent plant phosphoproteomics publications have identified several thousand phosphopeptides. However, the methods used in these studies are very labor-intensive and therefore not suitable to apply to a well-replicated circadian time series. To address this issue, we present and compare different strategies for sample preparation for phosphoproteomics that are compatible with large numbers of samples. Methods are compared regarding number of identifications, variability of quantitation, and functional categorization. We focus on the type of detergent used for protein extraction as well as methods for its removal. We also test a simple two-fraction separation of the protein extract.
authors: SynthSys and School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.
authors: Johanna
authors: J
authors: Krahmer
authors: SynthSys and School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.
authors: Matthew M
authors: MM
authors: Hindle
authors: SynthSys and School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.
authors: Sarah F
authors: SF
authors: Martin
authors: SynthSys and School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.
authors: Thierry
authors: T
authors: Le Bihan
authors: SynthSys and School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom. Electronic address: andrew.millar@ed.ac.uk.
authors: Andrew J
authors: AJ
authors: Millar
mesh_terms: D017360:Arabidopsis / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D057906:Circadian Clocks; D063990:Gene Ontology; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D029681:Arabidopsis Proteins; D010750:Phosphoproteins; D020543:Proteome
keywords: Arabidopsis; Circadian clock; Mass spectrometry; Phosphoproteomics; Protein extraction; Proteomics