The development of barley seed protein library for targeted analyses

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F62156489%3A43210%2F16%3A43911090" target="_blank" >RIV/62156489:43210/16:43911090 - isvavai.cz</a>

Výsledek na webu

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DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

The development of barley seed protein library for targeted analyses

Popis výsledku v původním jazyce

Proteomic analysis is probably the best approach to analyse seed germination. However, it is difficult to analyse complex samples and there are many obstacles that must be faced in order to achieve a reasonable proteome coverage. For example, the barley (Hordeum vulgare) genome was fully sequenced in 2012, but the UniProt database contains less than 1,000 reviewed sequences, which is approximately 16-fold less than for Arabidopsis thaliana. Here, to improve the barley proteome coverage, we employed several fractionation methods including polyethylene glycol precipitation, strong cation exchange chromatography, Off-Gel separation, SDS-PAGE and acetonitrile elution gradient. Proteomic analyses were performed using an LC-MS-based analyses and an UHR q-TOF mass spectrometer. The candidate peptides were targeted via Selected Reaction Monitoring (SRM) and Triple-stage quadrupole (TSQ) mass spectrometer. In total, 4,092 proteins were identified, which represents a three-to four-fold increase compared with the standard shotgun analysis of the same sample. Out of these, 2,240 were only accessible by one of the techniques and, besides, the detection limits were not similar. We hypothesized that an SRM-based targeted analysis will allow detection and quantitation of most of these proteins, even without the application of proteome fractionation. We can conclude that all peptides from the library with MS/MS spectra of the total intensity above 10,000 are easily detectable in the total protein extracts.

Název v anglickém jazyce

The development of barley seed protein library for targeted analyses

Popis výsledku anglicky

Proteomic analysis is probably the best approach to analyse seed germination. However, it is difficult to analyse complex samples and there are many obstacles that must be faced in order to achieve a reasonable proteome coverage. For example, the barley (Hordeum vulgare) genome was fully sequenced in 2012, but the UniProt database contains less than 1,000 reviewed sequences, which is approximately 16-fold less than for Arabidopsis thaliana. Here, to improve the barley proteome coverage, we employed several fractionation methods including polyethylene glycol precipitation, strong cation exchange chromatography, Off-Gel separation, SDS-PAGE and acetonitrile elution gradient. Proteomic analyses were performed using an LC-MS-based analyses and an UHR q-TOF mass spectrometer. The candidate peptides were targeted via Selected Reaction Monitoring (SRM) and Triple-stage quadrupole (TSQ) mass spectrometer. In total, 4,092 proteins were identified, which represents a three-to four-fold increase compared with the standard shotgun analysis of the same sample. Out of these, 2,240 were only accessible by one of the techniques and, besides, the detection limits were not similar. We hypothesized that an SRM-based targeted analysis will allow detection and quantitation of most of these proteins, even without the application of proteome fractionation. We can conclude that all peptides from the library with MS/MS spectra of the total intensity above 10,000 are easily detectable in the total protein extracts.

Druh

O - Ostatní výsledky

CEP obor

EB - Genetika a molekulární biologie

OECD FORD obor

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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

2016

Kód důvěrnosti údajů

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