Frakcionace obohacených fosfopeptidů s využittím separace na mikrokoloně pomocí pH/acetonitril-reverzně fázového gradientu v kombinaci s LC-MS/MS analýzou
| Autor: | Ales Tichy, Helena Rehulkova, Pavel Rehulka, Rudolf Kupcik, Martin Ondrej |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Phosphopeptides Proteomics enrichment Proteome Chemical Fractionation oxid titaničitý lcsh:Chemistry chemistry.chemical_compound hmotnostní spektrometrie Tandem Mass Spectrometry Phase (matter) lcsh:QH301-705.5 Spectroscopy mass spectrometry Titanium Chemistry Phosphopeptide Phosphoproteomics General Medicine Hydrogen-Ion Concentration Computer Science Applications gradient Acetonitriles Fractionation Mass spectrometry Catalysis obohacení Article Inorganic Chemistry 03 medical and health sciences Cell Line Tumor Lc ms ms Pressure Humans fractionation Physical and Theoretical Chemistry Acetonitrile Molecular Biology Chromatography acetonitril 030102 biochemistry & molecular biology frakcionace titanium dioxide Organic Chemistry fosfopeptidy Phosphoproteins acetonitrile 030104 developmental biology lcsh:Biology (General) lcsh:QD1-999 phosphopeptides Protein identification Chromatography Liquid |
| Zdroj: | International Journal of Molecular Sciences Volume 21 Issue 11 International Journal of Molecular Sciences, Vol 21, Iss 3971, p 3971 (2020) |
| ISSN: | 1422-0067 |
| Popis: | 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&ndash mass spectrometry/mass spectrometry (LC&ndash 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&ndash 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&ndash 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|