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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Benschop JJ, Mohammed S, O’Flaherty M, Heck AJ, Slijper M, Menke FL (2007) Quantitative phospho-proteomics of early elicitor signalling in Arabidopsis. Mol Cell Proteomics 6(7):1705–1713
Chen Y, Höhenwarter W, Weckwerth W (2010) Comparative analysis of phytohormone—responsive phosphoproteins in Arabidopsis thaliana using TiO2-phosphopeptide enrichment and MAPA. Plant J 63(1):1–17
Chinchilla D, Zipfel C, Robatzek S, Kemmerling B, Nurnberger T, Jones JD, Felix G, Boller T (2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448(7152):497–500. https://doi.org/10.1038/nature05999
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(12):1367–1372. https://doi.org/10.1038/nbt.1511
Engelsberger WR, Schulze WX (2012) Nitrate and ammonium lead to distinct global dynamic phosphorylation patterns when resupplied to nitrogen starved Arabidopsis seedlings. Plant J 69(6):978–995
Haruta M, Gray WM, Sussman MR (2015) Regulation of the plasma membrane proton pump (H(+)-ATPase) by phosphorylation. Curr Opin Plant Biol 28:68–75. https://doi.org/10.1016/j.pbi.2015.09.005
Kerk D, Bulgrien J, Smith DW, Barsam B, Veretnik S, Gribskov M (2002) The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis. Plant Physiol 129(2):908–925. https://doi.org/10.1104/pp.004002
Kim TW, Guan S, Sun Y, Deng Z, Tang W, Shang JX, Sun Y, Burlingam A, Wang ZY (2009) Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors. Nat Cell Biol 11(10):1254–1260
Lan P, Li W, Wen TN, Schmidt W (2012) Quantitative phosphoproteome profiling of iron-deficient Arabidopsis roots. Plant Physiol 159(1):403–417
Marshall A, Aalen RB, Audenaert D, Beeckman T, Broadley MR, Butenko MA, Caño-Delgado AI et al (2012) Tackling drought stress: receptor-like kinases present new approaches. Plant Cell 24(6):2262–2278
Menz J, Li Z, Schulze WX, Ludewig U (2016) Early nitrogen-deprivation responses in Arabidopsis roots reveal distinct differences on transcriptome and (phospho-) proteome levels between nitrate and ammonium nutrition. Plant J 88(5):717–734. https://doi.org/10.1111/tpj.13272
Niittylä T, Fuglsang AT, Palmgren MG, Frommer WB, Schulze WX (2007) Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis. Mol Cell Proteomics 6(10):1711–1726
Osakabe Y, Yamaguchi-Shinozaki K, Shinozaki K, Tran LS (2013) Sensing the environment: key roles of membrane-localized kinases in plant perception and response to abiotic stress. J Exp Bot 64(2):445–458
Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75(3):663–670
Reiland S, Finazzi G, Endler A, Willig A, Baerenfaller K, Grossmann J, Gerrits B et al (2011) Comparative phosphoproteome profiling reveals a function of the STN8 kinase in fine-tuning of cyclic electron flow (CEF). Proc Natl Acad Sci U S A 108(31):12955–12960
Reiland S, Messerli G, Baerenfäller K, Gerrits B, Endler A, Grossmann J, Gruissem W, Baginsky S (2009) Large-scale Arabidopsis phosphoproteome profiling reveals novel chloroplast kinase substrates and phosphorylation networks. Plant Physiol 150(2):889–903
Rudashevskaya EL, Ye J, Jensen ON, Fuglsang AT, Palmgren MG (2012) Phosphosite mapping of P-type plasma membrane H+-ATPase in homologous and heterologous environments. J Biol Chem 287(7):4904–4913
Ryu H, Kim K, Cho H, Hwang I (2010) Predominant actions of cytosolic BSU1 and nuclear BIN2 regulate subcellular localization of BES1 in brassinosteroid signaling. Mol Cells 29(3):291–296. https://doi.org/10.1007/s10059-010-0034-y
Schweighofer A, Meskiene I (2015) Phosphatases in plants. Methods Mol Biol 1306:25–46. https://doi.org/10.1007/978-1-4939-2648-0_2
Shiu SH, Bleecker AB (2003) Expansion of the receptor-like kinase/Pelle gene family and receptor-like proteins in Arabidopsis. Plant Physiol 132(2):530–543. https://doi.org/10.1104/pp.103.021964
Tran HT, Plaxton WC (2008) Proteomic analysis of alterations in the secretome of Arabidopsis thaliana suspension cells subjected to nutritional phosphate deficiency. Proteomics 8. https://doi.org/10.1002/pmic.200800292
Tyanova S, Temu T, Cox J (2016) The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11(12):2301–2319. https://doi.org/10.1038/nprot.2016.136
Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13(9):731–740. https://doi.org/10.1038/nmeth.3901
Wang R, Liu M, Yuan M, Oses-Prieto JA, Cai X, Sun Y, Burlingame AL, Wang ZY, Tang W (2016) The brassinosteroid-activated BRI1 receptor kinase is switched off by dephosphorylation mediated by cytoplasm-localized PP2A B’ subunits. Mol Plant 9(1):148–157. https://doi.org/10.1016/j.molp.2015.10.007
Wang X, Kota U, He K, Blackburn K, Li J, Goshe MB, Huber SC, Clouse SD (2008) Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling. Dev Cell 15(2):220–235. https://doi.org/10.1016/j.devcel.2008.06.011
Wang ZY, Nakano T, Gendron J, He J, Chen M, Vafeados D, Yang Y et al (2002) Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis. Dev Cell 2(4):505–513
Wu XN, Xi L, Pertl-Obermeyer H, Li Z, Chu LC, Schulze WX (2017) Highly efficient single-step enrichment of low abundance phosphopeptides from plant membrane preparations. Front Plant Sci 8:1673. https://doi.org/10.3389/fpls.2017.01673
Wu X, Sanchez-Rodriguez C, Pertl-Obermeyer H, Obermeyer G, Schulze WX (2013) Sucrose-induced receptor kinase SIRK1 regulates a plasma membrane aquaporin in Arabidopsis. Mol Cell Proteomics 12(10):2856–2873
Wu X, Sklodowski K, Encke B, Schulze WX (2014) A kinase-phosphatase signaling module with BSK8 and BSL2 involved in regulation of sucrose-phosphate synthase. J Proteome Res 13:3397–3409
Zhang H, Zhou H, Berke L, Heck AJ, Mohammed S, Scheres B, Menke FL (2013) Quantitative phosphoproteomics after auxin-stimulated lateral root induction identifies an SNX1 protein phosphorylation site required for growth. Mol Cell Proteomics 12(5):1158–1169
Zulawski M, Schulze G, Braginets R, Hartmann S, Schulze WX (2014) The Arabidopsis Kinome: phylogeny and evolutionary insights into functional diversification. BMC Genomics 15:548. https://doi.org/10.1186/1471-2164-15-548
Zulawski M, Schulze WX (2015) The plant kinome. Methods Mol Biol 1306:1–23. https://doi.org/10.1007/978-1-4939-2648-0_1
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
Xi, L., Schulze, W.X., Wu, X.N. (2021). Phosphoproteomic Analysis of Plant Membranes. In: Sanchez-Serrano, J.J., Salinas, J. (eds) Arabidopsis Protocols . Methods in Molecular Biology, vol 2200. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0880-7_22
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0880-7_22
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0879-1
Online ISBN: 978-1-0716-0880-7
eBook Packages: Springer Protocols