Abstract
Mass spectrometry (MS)-based large scale phosphoproteomics has facilitated the investigation of plant phosphorylation dynamics on a system-wide scale. However, generating large scale data sets for membrane phosphoproteins usually requires fractionation of samples and extended hands-on laboratory time. To overcome these limitations, we developed “ShortPhos,” an efficient and simple phosphoproteomics protocol optimized for research on plant membrane proteins. The optimized workflow allows fast and efficient identification and quantification of phosphopeptides, even from small amounts of starting plant materials. “ShortPhos” can produce label-free datasets with a high quantitative reproducibility. In addition, the “ShortPhos” protocol recovered more phosphorylation sites from membrane proteins, especially plasma membrane and vacuolar proteins, when compared to our previous workflow and other membrane-based data in the PhosPhAt 4.0 database. We applied “ShortPhos” to study kinase-substrate relationships within a nitrate-induction experiment on Arabidopsis roots. The “ShortPhos” identified significantly more known kinase-substrate relationships compared to previous phosphoproteomics workflows, producing new insights into nitrate-induced signaling pathways.
Highlights
Protein phosphorylation is regulated by protein kinases and phosphatases
We investigated the coverage of membrane proteins identified in the “ShortPhos” workflow compared to previously published data sets in PhosPhAt 4.0 database (Figure 5A)
Our “ShortPhos” workflow is based on three major improvements: Firstly, we applied an optimized microsomal fraction (MF) isolation method instead of traditional plasma membrane (PM) isolation, which saved much hands-on time and cost
Summary
Protein phosphorylation is regulated by protein kinases and phosphatases. These enzymes are key players in cellular signal transduction pathways catalyzing phosphorylation or dephosphorylation of target proteins and thereby modulating kinase signaling cascades (Hrabak et al, 2003; Pitzschke et al, 2009; Weinl and Kudla, 2009; Asano et al, 2012; Meng and Zhang, 2013), membrane transport (Liu and Tsay, 2003; Tornroth-Horsefield et al, 2006; Lanquar et al, 2009; Rudashevskaya et al, 2012; Fuglsang et al, 2014), and metabolic activities (Sanchez and Heldt, 1990; Mcmichael et al, 1993; Huber and Huber, 1996). Many protein kinases are regulated by autophosphorylation or by upstream kinases in signaling cascades (Zulawski et al, 2013; Zulawski and Schulze, 2015). Kinase substrates such as transporters, transcription factors or metabolic enzymes are modulated in response to external or internal stimuli and result in adaptive phenotypes (Czechowski et al, 2005; Chinchilla et al, 2007). The knowledge of kinase-substrate relationships is not complete, as for many kinases the substrates are still unknown, and for many substrates, the respective kinases are not known
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