Cargando…
Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC–MS/MS Analysis
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 h...
Autores principales: | , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312998/ https://www.ncbi.nlm.nih.gov/pubmed/32492839 http://dx.doi.org/10.3390/ijms21113971 |
_version_ | 1783549856931905536 |
---|---|
author | Ondrej, Martin Rehulka, Pavel Rehulkova, Helena Kupcik, Rudolf Tichy, Ales |
author_facet | Ondrej, Martin Rehulka, Pavel Rehulkova, Helena Kupcik, Rudolf Tichy, Ales |
author_sort | Ondrej, Martin |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-7312998 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-73129982020-06-29 Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC–MS/MS Analysis Ondrej, Martin Rehulka, Pavel Rehulkova, Helena Kupcik, Rudolf Tichy, Ales Int J Mol Sci Article 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. MDPI 2020-06-01 /pmc/articles/PMC7312998/ /pubmed/32492839 http://dx.doi.org/10.3390/ijms21113971 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Ondrej, Martin Rehulka, Pavel Rehulkova, Helena Kupcik, Rudolf Tichy, Ales Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC–MS/MS Analysis |
title | Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC–MS/MS Analysis |
title_full | Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC–MS/MS Analysis |
title_fullStr | Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC–MS/MS Analysis |
title_full_unstemmed | Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC–MS/MS Analysis |
title_short | Fractionation of Enriched Phosphopeptides Using pH/Acetonitrile-Gradient-Reversed-Phase Microcolumn Separation in Combination with LC–MS/MS Analysis |
title_sort | fractionation of enriched phosphopeptides using ph/acetonitrile-gradient-reversed-phase microcolumn separation in combination with lc–ms/ms analysis |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312998/ https://www.ncbi.nlm.nih.gov/pubmed/32492839 http://dx.doi.org/10.3390/ijms21113971 |
work_keys_str_mv | AT ondrejmartin fractionationofenrichedphosphopeptidesusingphacetonitrilegradientreversedphasemicrocolumnseparationincombinationwithlcmsmsanalysis AT rehulkapavel fractionationofenrichedphosphopeptidesusingphacetonitrilegradientreversedphasemicrocolumnseparationincombinationwithlcmsmsanalysis AT rehulkovahelena fractionationofenrichedphosphopeptidesusingphacetonitrilegradientreversedphasemicrocolumnseparationincombinationwithlcmsmsanalysis AT kupcikrudolf fractionationofenrichedphosphopeptidesusingphacetonitrilegradientreversedphasemicrocolumnseparationincombinationwithlcmsmsanalysis AT tichyales fractionationofenrichedphosphopeptidesusingphacetonitrilegradientreversedphasemicrocolumnseparationincombinationwithlcmsmsanalysis |