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

Result code in 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>

Alternative codes found

RIV/60162694:G44__/20:00555944

Result on the web

<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>

Result language

angličtina

Original language name

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

Original language description

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.

Czech name

—

Czech description

—

Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10406 - Analytical chemistry

Project

<a href="/en/project/NV19-02-00297" target="_blank" >NV19-02-00297: Proteomic analysis of the potential markers in patients with dilated cardiomyopathy</a><br>

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2020

Confidentiality

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

Name of the periodical

International Journal of Molecular Sciences

ISSN

1661-6596

e-ISSN

—

Volume of the periodical

21

Issue of the periodical within the volume

11

Country of publishing house

CH - SWITZERLAND

Number of pages

14

Pages from-to

3971

UT code for WoS article

000543400300233

EID of the result in the Scopus database

2-s2.0-85085895215