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 plantRamonda serbicais 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 toR.serbicaleaves, 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 desiccatedR.serbicaleaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the firstR.serbicaannotated transcriptome database, available at. The detergent-free phenol-based extraction combined with dode...cyl-beta-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-beta-D-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance inR.serbica, and we recommend this protocol for similar recalcitrant plant material.
Кључне речи:
Soluble and membrane-bound protein extraction / Resurrection plants / Recalcitrant plant material / Ramonda serbica / Phenol-based extraction / Peptide LC-MS / MS analysisИзвор:
Analytical and Bioanalytical Chemistry, 2020, 412, 30, 8299-8312Издавач:
- Springer Heidelberg, Heidelberg
Финансирање / пројекти:
- Министарство науке, технолошког развоја и иновација Републике Србије, институционално финансирање - 200053 (Универзитет у Београду, Институт за мултидисциплинарна истраживања) (RS-MESTD-inst-2020-200053)
- Science Fund of the Republic of Serbia (PROMIS project LEAPSyn-SCI) [6039663]
- University of Padova [BIRD189887/18]
- COST ActionEuropean Cooperation in Science and Technology (COST) [BM1405 (STSM-BM1405-190218-092344), BM1405 (STSM-BM1405-190317-080965)]
DOI: 10.1007/s00216-020-02965-2
ISSN: 1618-2642
PubMed: 33037906
WoS: 000578472500002
Scopus: 2-s2.0-85092359930
Институција/група
Institut za multidisciplinarna istraživanjaTY - JOUR AU - Vidović, Marija AU - Franchin, Cinzia AU - Morina, Filis AU - Veljović-Jovanović, Sonja AU - Masi, Antonio AU - Arrigoni, Giorgio PY - 2020 UR - http://rimsi.imsi.bg.ac.rs/handle/123456789/1322 AB - Resurrection plantRamonda serbicais 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 toR.serbicaleaves, 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 desiccatedR.serbicaleaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the firstR.serbicaannotated transcriptome database, available at. The detergent-free phenol-based extraction combined with dodecyl-beta-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-beta-D-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance inR.serbica, and we recommend this protocol for similar recalcitrant plant material. PB - Springer Heidelberg, Heidelberg T2 - Analytical and Bioanalytical Chemistry T1 - Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants EP - 8312 IS - 30 SP - 8299 VL - 412 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 plantRamonda serbicais 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 toR.serbicaleaves, 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 desiccatedR.serbicaleaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the firstR.serbicaannotated transcriptome database, available at. The detergent-free phenol-based extraction combined with dodecyl-beta-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-beta-D-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance inR.serbica, and we recommend this protocol for similar recalcitrant plant material.", publisher = "Springer Heidelberg, Heidelberg", journal = "Analytical and Bioanalytical Chemistry", title = "Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants", pages = "8312-8299", number = "30", volume = "412", 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 Springer Heidelberg, Heidelberg., 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 . .