Abstract
Mass spectrometry (MS)-based phosphoproteomics is a powerful tool for investigating cell signaling, yet it remains challenging to study plant phosphoproteomes due to the low yield of cell lysis and high complexity of plant lysate. Here we report a streamlined sample preparation workflow to analyze plant phosphoproteomes in a high-throughput manner. This workflow addresses the problem of low yield in the lysis step and eliminates the interferences of pigments and metabolites in plant lysate. Integrating chemical labeling and high pH reverse phase fractionation with this workflow achieves in-depth phosphoproteomic coverage. Notably, the scalability of this approach is demonstrated by systematically analyzing the effect of long-term cold stress in the perturbation of the tomato phosphoproteome. Identification of more than 30,000 phosphopeptides from tomato leaves and more than 5000 kinase-substrate pairs from Arabidopsis create the largest phosphoproteomic and signaling network resource to date.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Zhao C, Wang P, Si T et al (2017) MAP kinase cascades regulate the cold response by modulating ICE1 protein stability. Dev Cell 43(5):618–629
Wang P, Zhao Y, Li Z et al (2018) Reciprocal regulation of the TOR kinase and ABA receptor balances plant growth and stress response. Mol Cell 69(1):100–112
Lin Z, Li Y, Zhang Z et al (2020) A RAF-SnRK2 kinase cascade mediates early osmotic stress signaling in higher plants. Nat Commun 11(1):613. https://doi.org/10.1038/s41467-020-14477-9
Wang P, Xue L, Batelli G et al (2013) Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action. Proc Natl Acad Sci U S A 110(27):11205–11210
Umezawa T, Takahashi F, Shinozaki K (2014) Phosphorylation networks in the abscisic acid signaling pathway. Enzyme 35:27–56
Sugiyama N, Masuda T, Shinoda K et al (2007) Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications. Mol Cell Proteomics 6(6):1103–1109
Tsai CF, Hsu CC, Hung JN et al (2014) Sequential phosphoproteomic enrichment through complementary metal-directed immobilized metal ion affinity chromatography. Anal Chem 86(1):685–693
Hsu C-C, Zhu Y, Arrington JV et al (2018) Universal plant phosphoproteomics workflow and its application to tomato signaling in response to cold stress. Mol Cell Proteomics 17(10):2068–2080
Masuda T, Sugiyama N, Tomita M et al (2011) Microscale phosphoproteome analysis of 10,000 cells from human cancer cell lines. Anal Chem 83(20):7698–7703
Boersema PJ, Raijmakers R, Lemeer S et al (2009) Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat Protoc 4(4):484–494
Iliuk AB, Martin VA, Alicie BM et al (2010) In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion-functionalized soluble nanopolymers. Mol Cell Proteomics 9(10):2162–2172
Dimayacyac-Esleta BR, Tsai CF, Kitata RB et al (2015) Rapid high-pH reverse phase StageTip for sensitive small-scale membrane proteomic profiling. Anal Chem 87(24):12016–12023
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2(8):1896–1906
Wang P, Hsu CC, Du Y et al (2020) Mapping proteome-wide targets of protein kinases in plant stress responses. Proc Natl Acad Sci U S A 117(6):3270–3280
Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372
Tyanova S, Temu T, Sinitcyn P et al (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13:731–740
Acknowledgments
The authors are grateful for financial support of this work from the NSF (grant 1506752).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Hsu, CC., Arrington, J.V., Tao, W.A. (2021). Universal Sample Preparation Workflow for Plant Phosphoproteomic Profiling. In: Wu, X.N. (eds) Plant Phosphoproteomics. Methods in Molecular Biology, vol 2358. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1625-3_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1625-3_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1624-6
Online ISBN: 978-1-0716-1625-3
eBook Packages: Springer Protocols