Chapter 22
Phosphoproteomic Analysis of Plant Membranes
Lin Xi, Waltraud X. Schulze, and Xu Na Wu
Abstract
Mass spectrometry (MS) is a powerful tool to investigate plant phosphorylation dynamics on a system-wide
scale (phosphoproteomics). Plant membrane phosphoproteomics enables elucidating regulatory patterns in
membranes, such as kinase-target relationships in different signaling pathways. Here, we present “ShortPhos,” an efficient and simple phosphoproteomics protocol for research on plant membrane proteins,
which allows fast and efficient identification and quantification of phosphopeptides from small amounts of
starting plant material and/or membrane proteins. This method improves upon the efficiency of plant
membrane phosphoproteomics profiling and can be applied to the study of membrane-based signaling
networks.
Key words Plant membrane proteins, Phosphoproteomics, Mass spectrometry
1 Introduction
Protein phosphorylation is a widespread posttranslational modification that regulates cellular signaling processes. This modification
is conducted by protein kinases [31, 32] and the phosphate group
is generally removed by phosphatases [19]. Mass spectrometry
(MS)-based phosphoproteomics has been used to study the dynamics of global phosphorylation for more than a decade in plants in the
context of nutrient stimulation [5, 9, 11, 12, 21, 28, 29], defense
[1], primary metabolism [15, 16], and hormone signaling [2, 30].
Membrane proteins possess crucial functions in membranebased signaling pathways [7, 20, 24, 31]. For example, receptor
kinases have key roles in the perception of external signals and in
regulation of signal transduction by phosphorylation [3, 8, 10, 13].
Brassinolide signal transduction has been well studied in the past
years. The BRI1/BAK1 receptor complex phosphorylates each
other sequentially to completely activate BRI1, and then BRI1 is
able to phosphorylate downstream targets (e.g., BSU1, BZR1)
[18, 24–26]. Using a plant membrane phosphoproteomics
approach, we identified the phosphorylation target of receptor
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_22, © Springer Science+Business Media, LLC, part of Springer Nature 2021
441
Phosphoproteomic Analysis of Plant Membranes
Lin Xi, Waltraud X. Schulze, and Xu Na Wu
Abstract
Mass spectrometry (MS) is a powerful tool to investigate plant phosphorylation dynamics on a system-wide
scale (phosphoproteomics). Plant membrane phosphoproteomics enables elucidating regulatory patterns in
membranes, such as kinase-target relationships in different signaling pathways. Here, we present “ShortPhos,” an efficient and simple phosphoproteomics protocol for research on plant membrane proteins,
which allows fast and efficient identification and quantification of phosphopeptides from small amounts of
starting plant material and/or membrane proteins. This method improves upon the efficiency of plant
membrane phosphoproteomics profiling and can be applied to the study of membrane-based signaling
networks.
Key words Plant membrane proteins, Phosphoproteomics, Mass spectrometry
1 Introduction
Protein phosphorylation is a widespread posttranslational modification that regulates cellular signaling processes. This modification
is conducted by protein kinases [31, 32] and the phosphate group
is generally removed by phosphatases [19]. Mass spectrometry
(MS)-based phosphoproteomics has been used to study the dynamics of global phosphorylation for more than a decade in plants in the
context of nutrient stimulation [5, 9, 11, 12, 21, 28, 29], defense
[1], primary metabolism [15, 16], and hormone signaling [2, 30].
Membrane proteins possess crucial functions in membranebased signaling pathways [7, 20, 24, 31]. For example, receptor
kinases have key roles in the perception of external signals and in
regulation of signal transduction by phosphorylation [3, 8, 10, 13].
Brassinolide signal transduction has been well studied in the past
years. The BRI1/BAK1 receptor complex phosphorylates each
other sequentially to completely activate BRI1, and then BRI1 is
able to phosphorylate downstream targets (e.g., BSU1, BZR1)
[18, 24–26]. Using a plant membrane phosphoproteomics
approach, we identified the phosphorylation target of receptor
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_22, © Springer Science+Business Media, LLC, part of Springer Nature 2021
441