Sample preparation for phosphoproteomic analysis of circadian time series in Arabidopsis thaliana

Johanna Krahmer, Matthew M Hindle, Sarah F Martin, Thierry Le Bihan, Andrew J Millar*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

Abstract / Description of output

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.

Original languageEnglish
Title of host publicationMethods in Enzymology
Subtitle of host publicationMembrane Proteins—Engineering, Purification and Crystallization
Number of pages27
ISBN (Print)978-0-12-802183-5
Publication statusPublished - 2015

Keywords / Materials (for Non-textual outputs)

  • Arabidopsis
  • Circadian clock
  • Mass spectrometry
  • Phosphoproteomics
  • Protein extraction
  • Proteomics


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