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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60077344%3A_____%2F20%3A00541294" target="_blank" >RIV/60077344:_____/20:00541294 - isvavai.cz</a>

Výsledek na webu

<a href="https://link.springer.com/article/10.1007%2Fs00216-020-02965-2" target="_blank" >https://link.springer.com/article/10.1007%2Fs00216-020-02965-2</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1007/s00216-020-02965-2" target="_blank" >10.1007/s00216-020-02965-2</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2020

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Analytical and Bioanalytical Chemistry

ISSN

1618-2642

e-ISSN

—

Svazek periodika

412

Číslo periodika v rámci svazku

30

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

14

Strana od-do

8299-8312

Kód UT WoS článku

000578472500002

EID výsledku v databázi Scopus

2-s2.0-85092359930