Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC-MS/MS Analysis

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216275%3A25310%2F20%3A39916593" target="_blank" >RIV/00216275:25310/20:39916593 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/60162694:G44__/20:00555944

Výsledek na webu

<a href="https://www.mdpi.com/1422-0067/21/11/3971/htm" target="_blank" >https://www.mdpi.com/1422-0067/21/11/3971/htm</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3390/ijms21113971" target="_blank" >10.3390/ijms21113971</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC-MS/MS Analysis

Popis výsledku v původním jazyce

Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.

Název v anglickém jazyce

Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC-MS/MS Analysis

Popis výsledku anglicky

Mass spectrometry (MS) is a powerful and sensitive method often used for the identification of phosphoproteins. However, in phosphoproteomics, there is an identified need to compensate for the low abundance, insufficient ionization, and suppression effects of non-phosphorylated peptides. These may hamper the subsequent liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, resulting in incomplete phosphoproteome characterization, even when using high-resolution instruments. To overcome these drawbacks, we present here an effective microgradient chromatographic technique that yields specific fractions of enriched phosphopeptides compatible with LC-MS/MS analysis. The purpose of our study was to increase the number of identified phosphopeptides, and thus, the coverage of the sample phosphoproteome using the reproducible and straightforward fractionation method. This protocol includes a phosphopeptide enrichment step followed by the optimized microgradient fractionation of enriched phosphopeptides and final LC-MS/MS analysis of the obtained fractions. The simple fractionation system consists of a gas-tight microsyringe delivering the optimized gradient mobile phase to reversed-phase microcolumn. Our data indicate that combining the phosphopeptide enrichment with the microgradient separation is a promising technique for in-depth phosphoproteomic analysis due to moderate input material requirements and more than 3-fold enhanced protein identification.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10406 - Analytical chemistry

Projekt

<a href="/cs/project/NV19-02-00297" target="_blank" >NV19-02-00297: Proteomická analýza potenciálních markerů dilatační kardiomyopatie</a><br>

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

International Journal of Molecular Sciences

ISSN

1661-6596

e-ISSN

—

Svazek periodika

21

Číslo periodika v rámci svazku

11

Stát vydavatele periodika

CH - Švýcarská konfederace

Počet stran výsledku

14

Strana od-do

3971

Kód UT WoS článku

000543400300233

EID výsledku v databázi Scopus

2-s2.0-85085895215