The development of barley seed protein library for targeted analyses

Result code in 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>

Result on the web

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DOI - Digital Object Identifier

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Result language

angličtina

Original language name

The development of barley seed protein library for targeted analyses

Original language description

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.

Czech name

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Czech description

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Type

O - Miscellaneous

CEP classification

EB - Genetics and molecular biology

OECD FORD branch

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Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

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

Publication year

2016

Confidentiality

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