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Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants

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Abstract

Resurrection plant Ramonda serbica is a suitable model to investigate vegetative desiccation tolerance. However, the detailed study of these mechanisms at the protein level is hampered by the severe tissue water loss, high amount of phenolics and polysaccharide, and possible protein modifications and aggregations during the extraction and purification steps. When applied to R. serbica leaves, widely used protein extraction protocols containing polyvinylpolypyrrolidone and ascorbate, as well as the phenol/SDS/buffer–based protocol recommended for recalcitrant plant tissues failed to eliminate persistent contamination and ensure high protein quality. Here we compared three protein extraction approaches aiming to establish the optimal one for both hydrated and desiccated R. serbica leaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the first R. serbica annotated transcriptome database, available at http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta. The detergent-free phenol-based extraction combined with dodecyl-β-d-maltoside-assisted extraction enabled high-yield and high-purity protein extracts. The phenol-based protocol improved the protein-band resolution, band number, and intensity upon electrophoresis, and increased the protein yield and the number of identified peptides and protein groups by LC-MS/MS. Additionally, dodecyl-β-d-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance in R. serbica, and we recommend this protocol for similar recalcitrant plant material.

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Data availability

R. serbica transcriptome database, publically available at http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta

R. serbica protein fasta file: http://www.biomed.unipd.it/filearrigoni/Ramonda_Serbica_Protein_fasta_file.fasta

Abbreviations

βME:

2-Mercaptoethanol

CHAPS:

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulphonate

DDM:

Dodecyl-β-d-maltoside

DL:

Desiccated leaves

EDTA:

Ethylenediaminetetraacetic acid

FASP:

Filter-aided sample preparation

HL:

Fully hydrated leaves

LC:

Liquid chromatography

MS:

Mass spectrometry

PAGE:

Polyacrylamide gel electrophoresis

PETC:

Photosynthetic electron transport chain

PMSF:

Phenylmethylsulphonyl fluoride

PS:

Photosystem

PSM:

Peptide-spectrum match

PVPP:

Water-insoluble polyvinylpolypyrrolidone

RWC:

Relative water content

SDS:

Sodium dodecyl sulphate

TCA:

Trichloroacetic acid

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Acknowledgements

The authors would like to acknowledge the technical help provided Dr. Anna Rita Trentin (DAFNAE, University of Padova) and Ilaria Battisti (Proteomics Center University of Padova and Azienda Ospedaliera di Padova). The authors wish to thank the Cassa di Risparmio di Padova e Rovigo (Cariparo) Holding for funding the acquisition of the LTQ-Orbitrap XL mass spectrometer.

Funding

This work was funded by the Ministry of Education, Science and Technological Development, Republic of Serbia (contract no. 451-03-68/2020-14/200053), the Science Fund of the Republic of Serbia (PROMIS project LEAPSyn-SCI, grant number 6039663), and the University of Padova (grant number BIRD189887/18 to G.A). M.Vidović received financial support from COST Action BM1405 (STSM-BM1405-190218-092344 and STSM-BM1405-190317-080965).

Author information

Author notes

  1. Marija Vidović

    Present address: Institute of Molecular Genetics and Genetic Engineering, Laboratory for Plant Molecular Biology, University of Belgrade, Vojvode Stepe 444a, Belgrade, 11042, Serbia

  2. Filis Morina

    Present address: Department of Plant Biophysics & Biochemistry, Biology Centre, Institute of Plant Molecular Biology, Czech Academy of Sciences, Branišovská 31/1160, 37005, České Budějovice, Czech Republic

Authors and Affiliations

  1. Institute for Multidisciplinary Research, Department of Life Science, University of Belgrade, Kneza Višeslava 1, Belgrade, 11030, Serbia

    Marija Vidović, Filis Morina & Sonja Veljović-Jovanović

  2. Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131, Padua, Italy

    Cinzia Franchin & Giorgio Arrigoni

  3. Proteomics Center University of Padova and Azienda Ospedaliera di Padova, Via G. Orus 2/B, 35129, Padua, Italy

    Cinzia Franchin & Giorgio Arrigoni

  4. DAFNAE - University of Padova, Viale Università 16 – AGRIPOLIS, I-35020, Legnaro, PD, Italy

    Antonio Masi

  5. CRIBI Biotechnology Center, University of Padova, viale G. Colombo 3, 35131, Padua, Italy

    Giorgio Arrigoni

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Contributions

M. Vidović and G. Arrigoni designed the research. F. Morina, S. Veljović-Jovanović, and M.Vidović collected the material and conducted the experiment. F. Morina extracted and purified total RNA. M. Vidović tested the protein extraction procedures. M. Vidović, C. Franchin, and G. Arrigoni performed peptide digestion, purification, and proteomic analysis. M. Vidović and G. Arrigoni analysed the data, and together with F. Morina, A. Masi, and S. Veljović-Jovanović discussed the results and wrote the manuscript. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Marija Vidović or Giorgio Arrigoni.

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Vidović, M., Franchin, C., Morina, F. et al. Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants. Anal Bioanal Chem 412, 8299–8312 (2020). https://doi.org/10.1007/s00216-020-02965-2

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