Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants
Само за регистроване кориснике
2020
Аутори
Vidović, MarijaFranchin, Cinzia
Morina, Filis
Veljović-Jovanović, Sonja
Masi, Antonio
Arrigoni, Giorgio
Чланак у часопису (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
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_Sam...ple_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.
Кључне речи:
Chromatography, Liquid / Desiccation / Electrophoresis, Polyacrylamide Gel / Magnoliopsida / Peptide LC-MS/MS analysis / Phenol-based extraction / Plant Leaves / Plant Proteins / Proteomics / Ramonda serbica / Recalcitrant plant material / Resurrection plants / Soluble and membrane-bound protein extraction / Tandem Mass Spectrometry / WaterИзвор:
Analytical and Bioanalytical Chemistry, 2020, 412, 30, 8299-8312Финансирање / пројекти:
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200053 (Универзитет у Београду, Институт за мултидисциплинарна истраживања) (RS-200053)
- LEAPSyn-SCI - Late Embryogenesis Abundant Proteins: Structural Characterisation and Interaction With Α-Synuclein (RS-6039663)
- University of Padova (grant number BIRD189887/18 to G.A.
- COST Action BM1405 (STSM-BM1405-190218-092344 and STSM-BM1405-190317- 080965).
Напомена:
- Supplementary material: https://cherry.chem.bg.ac.rs/handle/123456789/4504
Повезане информације:
DOI: 10.1007/s00216-020-02965-2
ISSN: 1618-2650
WoS: 000578472500002
Scopus: 2-s2.0-85092359930
Колекције
Институција/група
Hemijski fakultet / Faculty of ChemistryTY - JOUR AU - Vidović, Marija AU - Franchin, Cinzia AU - Morina, Filis AU - Veljović-Jovanović, Sonja AU - Masi, Antonio AU - Arrigoni, Giorgio PY - 2020 UR - http://www.ncbi.nlm.nih.gov/pubmed/33037906 UR - https://cherry.chem.bg.ac.rs/handle/123456789/4505 AB - 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. T2 - Analytical and Bioanalytical Chemistry T1 - Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants VL - 412 IS - 30 SP - 8299 EP - 8312 DO - 10.1007/s00216-020-02965-2 ER -
@article{ author = "Vidović, Marija and Franchin, Cinzia and Morina, Filis and Veljović-Jovanović, Sonja and Masi, Antonio and Arrigoni, Giorgio", year = "2020", 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.", journal = "Analytical and Bioanalytical Chemistry", title = "Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants", volume = "412", number = "30", pages = "8299-8312", doi = "10.1007/s00216-020-02965-2" }
Vidović, M., Franchin, C., Morina, F., Veljović-Jovanović, S., Masi, A.,& Arrigoni, G.. (2020). Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants. in Analytical and Bioanalytical Chemistry, 412(30), 8299-8312. https://doi.org/10.1007/s00216-020-02965-2
Vidović M, Franchin C, Morina F, Veljović-Jovanović S, Masi A, Arrigoni G. Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants. in Analytical and Bioanalytical Chemistry. 2020;412(30):8299-8312. doi:10.1007/s00216-020-02965-2 .
Vidović, Marija, Franchin, Cinzia, Morina, Filis, Veljović-Jovanović, Sonja, Masi, Antonio, Arrigoni, Giorgio, "Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants" in Analytical and Bioanalytical Chemistry, 412, no. 30 (2020):8299-8312, https://doi.org/10.1007/s00216-020-02965-2 . .
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