SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F21%3A00121574" target="_blank" >RIV/00216224:14740/21:00121574 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.frontiersin.org/articles/10.3389/fpls.2021.635550/full" target="_blank" >https://www.frontiersin.org/articles/10.3389/fpls.2021.635550/full</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3389/fpls.2021.635550" target="_blank" >10.3389/fpls.2021.635550</a>

Jazyk výsledku

angličtina

Název v původním jazyce

SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS

Popis výsledku v původním jazyce

Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.

Název v anglickém jazyce

SP3 Protocol for Proteomic Plant Sample Preparation Prior LC-MS/MS

Popis výsledku anglicky

Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10611 - Plant sciences, botany

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2021

Kód důvěrnosti údajů

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

Název periodika

Frontiers in Plant Science

ISSN

1664-462X

e-ISSN

—

Svazek periodika

12

Číslo periodika v rámci svazku

MAR

Stát vydavatele periodika

CH - Švýcarská konfederace

Počet stran výsledku

12

Strana od-do

„635550“

Kód UT WoS článku

000631851900001

EID výsledku v databázi Scopus

2-s2.0-85103216802