Proteomics-based identification of low-abundance signaling and regulatory protein complexes in native plant tissues

Owing to the low abundance of signaling proteins and transcription factors, their protein complexes are not easily identified by classical proteomics. The isolation of these protein complexes from endogenous plant tissues (rather than plant cell cultures) is therefore an important technical challenge. Here, we describe a sensitive, quantitative proteomics-based procedure to determine the composition of plant protein complexes. The method makes use of fluorophore-tagged protein immunoprecipitation (IP) and label-free mass spectrometry (MS)-based quantification to correct for nonspecifically precipitated proteins. We provide procedures for the isolation of membrane-bound receptor complexes and transcriptional regulators from nuclei. The protocol consists of an IP step (∼6 h) and sample preparation for liquid chromatography-tandem MS (LC-MS/MS; 2 d). We also provide a guide for data analysis. Our single-step affinity purification protocol is a good alternative to two-step tandem affinity purification (TAP), as it is shorter and relatively easy to perform. The data analysis by label-free quantification (LFQ) requires a cheaper and less challenging experimental setup compared with known labeling techniques in plants.

[1]  M. Fromm,et al.  Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants. , 2004, The Plant journal : for cell and molecular biology.

[2]  Heribert Hirt,et al.  Isolation and characterization of plant protein complexes by mass spectrometry , 2011, Proteomics.

[3]  C. Jonak,et al.  SPEECHLESS integrates brassinosteroid and stomata signalling pathways , 2012, Nature Cell Biology.

[4]  Charles Darwin,et al.  Experiments , 1800, The Medical and physical journal.

[5]  S. D. de Vries,et al.  Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase 1 protein is present in sporophytic and gametophytic cells and undergoes endocytosis , 2005, Protoplasma.

[6]  Rune Matthiesen,et al.  Stable Isotope Labeling of Arabidopsis thaliana Cells and Quantitative Proteomics by Mass Spectrometry*S , 2005, Molecular & Cellular Proteomics.

[7]  Wade H. Dunham,et al.  Affinity‐purification coupled to mass spectrometry: Basic principles and strategies , 2012, Proteomics.

[8]  U. Grossniklaus,et al.  The Arabidopsis Somatic Embryogenesis Receptor Kinase 1 Gene Is Expressed in Developing Ovules and Embryos and Enhances Embryogenic Competence in Culture , 2001 .

[9]  Lukas N. Mueller,et al.  An integrated mass spectrometric and computational framework for the analysis of protein interaction networks , 2007, Nature Biotechnology.

[10]  V. Rubio,et al.  An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation. , 2005, The Plant journal : for cell and molecular biology.

[11]  A. Hyman,et al.  Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions , 2010, The Journal of cell biology.

[12]  M. Selbach,et al.  Global quantification of mammalian gene expression control , 2011, Nature.

[13]  Shujing Liu,et al.  Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development , 2012, Proceedings of the National Academy of Sciences.

[14]  A. Nesvizhskii,et al.  Comparative analysis of different label-free mass spectrometry based protein abundance estimates and their correlation with RNA-Seq gene expression data. , 2012, Journal of proteome research.

[15]  Jürgen Cox,et al.  A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics , 2009, Nature Protocols.

[16]  Sara Linse,et al.  Methods for the detection and analysis of protein–protein interactions , 2007, Proteomics.

[17]  Michael Gribskov,et al.  Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice. , 2006, The Plant journal : for cell and molecular biology.

[18]  Martin R Larsen,et al.  Chemical deamidation: a common pitfall in large-scale N-linked glycoproteomic mass spectrometry-based analyses. , 2012, Journal of proteome research.

[19]  G. Theißen,et al.  The class E floral homeotic protein SEPALLATA3 is sufficient to loop DNA in ‘floral quartet’-like complexes in vitro , 2008, Nucleic acids research.

[20]  Jeffrey W. Smith,et al.  Mass Spectrometry-Based Label-Free Quantitative Proteomics , 2009, Journal of biomedicine & biotechnology.

[21]  Blagoy Blagoev,et al.  A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling , 2003, Nature Biotechnology.

[22]  Martin Kuiper,et al.  Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana , 2010, Molecular systems biology.

[23]  S. Clough,et al.  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[24]  C. Smaczniak,et al.  Proteomics insights into plant signaling and development , 2011, Proteomics.

[25]  Heinz Saedler,et al.  Plant biology: Floral quartets , 2001, Nature.

[26]  S. Henikoff,et al.  The INTACT method for cell type–specific gene expression and chromatin profiling in Arabidopsis thaliana , 2011, Nature Protocols.

[27]  Mikhail M Savitski,et al.  ModifiComb, a New Proteomic Tool for Mapping Substoichiometric Post-translational Modifications, Finding Novel Types of Modifications, and Fingerprinting Complex Protein Mixtures* , 2006, Molecular & Cellular Proteomics.

[28]  Antoine H P America,et al.  Comparative LC‐MS: A landscape of peaks and valleys , 2008, Proteomics.

[29]  E. Birney,et al.  The International Protein Index: An integrated database for proteomics experiments , 2004, Proteomics.

[30]  M. Mann,et al.  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification , 2008, Nature Biotechnology.

[31]  U. Grossniklaus,et al.  A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta[w] , 2003, Plant Physiology.

[32]  K. Resing,et al.  Comparison of Label-free Methods for Quantifying Human Proteins by Shotgun Proteomics*S , 2005, Molecular & Cellular Proteomics.

[33]  Eugenia Russinova,et al.  The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 Protein Complex Includes BRASSINOSTEROID-INSENSITIVE1[W] , 2006, The Plant Cell Online.

[34]  Jiří Friml,et al.  Cell Plate Restricted Association of DRP1A and PIN Proteins Is Required for Cell Polarity Establishment in Arabidopsis , 2011, Current Biology.

[35]  T. Veenstra,et al.  Proteomic analysis of protein complexes , 2007, Proteomics.

[36]  Kerstin Kaufmann,et al.  The 'ABC' of MADS domain protein behaviour and interactions. , 2010, Seminars in cell & developmental biology.

[37]  Karl Mechtler,et al.  BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals , 2008, Nature Methods.

[38]  M. Mann,et al.  Andromeda: a peptide search engine integrated into the MaxQuant environment. , 2011, Journal of proteome research.

[39]  R. Karlova.  The SERK1 protein complexes , 2008 .