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

Nature Protocols volume 7, pages 2144–2158 (2012)Cite this article

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Abstract

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.

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Figure 1: Experimental and simplified data analysis workflow.
Figure 2: Confocal images of the fluorophore-tagged proteins in native plant tissues.
Figure 3: Determination of the binding capacity and affinity of the anti-GFP beads.
Figure 4: Protein interaction profiling using MaxQuant.

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Acknowledgements

C.S. and G.C.A. were supported by a Proteomics Hotel grant from the Netherlands Proteomics Centre (NPCII-T2.3) and Centre for BioSystems Genomics (CBSG2012). All proteomic measurements were recorded at Biqualys Wageningen (http://www.biqualys.nl). N.L. was supported by the Netherlands Organization for Science, ALW grant no. 61.62.5000.86. N.L., W.v.D. and S.d.V. thank B. Möller for sharing details of IP protocols and helpful discussions. K.K. thanks M.C. O'Flaherty and R. Karlova for advice during initial stages of the project, as well as J.H. Cordewener and J.J.M. Vervoort for useful discussions. We thank J. Muiño for support in bioinformatics and statistical analysis.

Author information

Author notes

  1. Na Li

    Present address: Present address: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.,

  2. Cezary Smaczniak and Na Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Plant Research International,Business Unit Bioscience, Wageningen, The Netherlands

    Cezary Smaczniak, Twan America, Gerco C Angenent & Kerstin Kaufmann

  2. Laboratory of Molecular Biology, Wageningen University, Wageningen, The Netherlands

    Cezary Smaczniak & Kerstin Kaufmann

  3. Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands

    Na Li, Sjef Boeren, Walter van Dongen & Sacco de Vries

  4. Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research, Utrecht University,, Utrecht, The Netherlands

    Soenita S Goerdayal

  5. Netherlands Proteomics Centre (NPC), Utrecht, The Netherlands

    Soenita S Goerdayal

  6. Centre for BioSystems Genomics (CBSG), Wageningen, The Netherlands

    Gerco C Angenent

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  1. Cezary Smaczniak
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Contributions

C.S., N.L. and K.K. designed the research; C.S., N.L. and W.v.D. performed experiments; S.B. recorded LC-MS/MS measurements; C.S. and S.B. analyzed the data; S.B., T.A., S.d.V. and G.C.A. commented on the manuscript; S.S.G. provided trypsin digestion and peptide purification protocols for soluble proteins; K.K. supervised the project; C.S., N.L. and K.K. wrote the manuscript.

Corresponding author

Correspondence to Kerstin Kaufmann.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Methods for Label-free quantification with Progenesis LC-MS; Bimolecular fluorescence complementation (BiFC) and Fluorescence Resonance Energy Transfer-Fluorescence Lifetime Imaging Microscopy (FRET-FLIM). (PDF 274 kb)

Supplementary Note

A small database of sequences for background proteins that we expect to find in all samples. These are present in FASTA format to perform the first search. (TXT 32 kb)

Supplementary Data 1

MaxQuant analysis results of the PI-GFP and Col-0 immunoprecipitates: (A) MaxQuant output (proteinGroups.txt) dataset of protein quantification; (B) Statistical analysis with Perseus of the proteinGroups.txt dataset according to Steps 53-62 of the protocol; (C) Input parameters for MaxQuant; (D) Experimental design input table. The results presented here are based on the same raw data used in REF. 11. (XLSX 368 kb)

Supplementary Data 2

MaxQuant analysis results of the SERK1-CFP and Col-0 immunoprecipitates: (A) MaxQuant output (proteinGroups.txt) dataset of protein quantification; (B) Statistical analysis with Perseus of the proteinGroups.txt dataset according to Steps 53-62 of the protocol; (C) Input parameters for MaxQuant; (D) Experimental design input table. (XLSX 302 kb)

Supplementary Figure 1

Protein interaction profiling using Progenesis LC-MS on the example of PI-GFP IPs. Graphical representation of the normalized protein abundance ratios between the PI-GFP IP samples and controls plotted against total protein normalized abundance (summed peptide MS1 peak abundances – areas under peak). Imputation of the missing values with the lowest normalized protein abundance value. (PDF 938 kb)

Supplementary Figure 2

Interactions of SERK1 with At1g27190, At2g41820 and At3g28450 confirmed by FRET-FLIM and BiFC in Arabidopsis protoplasts. (A-C) Fluorescence lifetime images of A. thaliana leaf protoplasts transiently expressing SERK1-sCFP (A), SERK1-sCFP + At2g41820-sYFP (B) and SERK1-sCFP + At1g27190-sYFP (C) for 16 hrs. sCFP lifetime distributions are presented as pseudocolor images; the color bar shows the lifetime distribution, ranging from t = 2.0 ns (red) – 3.0 ns (blue). Average lifetimes, determined on at least 45 protoplasts in three independent experiments, are 2.62 ± 0.06 ns for SERK1-sCFP, 2.37 ± 0.06 ns for SERK1-sCFP/At1g27190-sYFP and 2.46 ± 0.09ns for SERK1-sCFP/At2g41820-sYFP. (D-F) Bimolecular fluorescence complementation of SERK1-YFPC with At1g27190-YFPN (D), At2g41820-YFPN (E) and At3g28450-YFPN (F) expressed in Arabidopsis leaf protoplasts for 16 hrs. (PDF 1516 kb)

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Smaczniak, C., Li, N., Boeren, S. et al. Proteomics-based identification of low-abundance signaling and regulatory protein complexes in native plant tissues. Nat Protoc 7, 2144–2158 (2012). https://doi.org/10.1038/nprot.2012.129

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