Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome

Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome

Abstract Protein phosphorylation is involved in the regulation of most eukaryotic cells functions and mass spectrometry-based analysis has made major contributions to our understanding of this regulation. However, low abundance of phosphorylated species presents a major challenge in achieving compre...

Descripción completa

Guardado en:
Detalles Bibliográficos
Autores principales: Elena Panizza, Rui M. M. Branca, Peter Oliviusson, Lukas M. Orre, Janne Lehtiö
Formato: article
Lenguaje:EN
Publicado: Nature Portfolio 2017
Materias:
Medicine
R
Science
Q
Acceso en línea:https://doaj.org/article/490425e62f0e446b872c186b12ec9123
Etiquetas: Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
id oai:doaj.org-article:490425e62f0e446b872c186b12ec9123
record_format dspace
spelling oai:doaj.org-article:490425e62f0e446b872c186b12ec91232021-12-02T15:06:18ZIsoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome10.1038/s41598-017-04798-z2045-2322https://doaj.org/article/490425e62f0e446b872c186b12ec91232017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-04798-zhttps://doaj.org/toc/2045-2322Abstract Protein phosphorylation is involved in the regulation of most eukaryotic cells functions and mass spectrometry-based analysis has made major contributions to our understanding of this regulation. However, low abundance of phosphorylated species presents a major challenge in achieving comprehensive phosphoproteome coverage and robust quantification. In this study, we developed a workflow employing titanium dioxide phospho-enrichment coupled with isobaric labeling by Tandem Mass Tags (TMT) and high-resolution isoelectric focusing (HiRIEF) fractionation to perform in-depth quantitative phosphoproteomics starting with a low sample quantity. To benchmark the workflow, we analyzed HeLa cells upon pervanadate treatment or cell cycle arrest in mitosis. Analyzing 300 µg of peptides per sample, we identified 22,712 phosphorylation sites, of which 19,075 were localized with high confidence and 1,203 are phosphorylated tyrosine residues, representing 6.3% of all detected phospho-sites. HiRIEF fractions with the most acidic isoelectric points are enriched in multiply phosphorylated peptides, which represent 18% of all the phospho-peptides detected in the pH range 2.5–3.7. Cross-referencing with the PhosphoSitePlus database reveals 1,264 phosphorylation sites that have not been previously reported and kinase association analysis suggests that a subset of these may be functional during the mitotic phase.Elena PanizzaRui M. M. BrancaPeter OliviussonLukas M. OrreJanne LehtiöNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-13 (2017)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Elena Panizza
Rui M. M. Branca
Peter Oliviusson
Lukas M. Orre
Janne Lehtiö
Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome
description Abstract Protein phosphorylation is involved in the regulation of most eukaryotic cells functions and mass spectrometry-based analysis has made major contributions to our understanding of this regulation. However, low abundance of phosphorylated species presents a major challenge in achieving comprehensive phosphoproteome coverage and robust quantification. In this study, we developed a workflow employing titanium dioxide phospho-enrichment coupled with isobaric labeling by Tandem Mass Tags (TMT) and high-resolution isoelectric focusing (HiRIEF) fractionation to perform in-depth quantitative phosphoproteomics starting with a low sample quantity. To benchmark the workflow, we analyzed HeLa cells upon pervanadate treatment or cell cycle arrest in mitosis. Analyzing 300 µg of peptides per sample, we identified 22,712 phosphorylation sites, of which 19,075 were localized with high confidence and 1,203 are phosphorylated tyrosine residues, representing 6.3% of all detected phospho-sites. HiRIEF fractions with the most acidic isoelectric points are enriched in multiply phosphorylated peptides, which represent 18% of all the phospho-peptides detected in the pH range 2.5–3.7. Cross-referencing with the PhosphoSitePlus database reveals 1,264 phosphorylation sites that have not been previously reported and kinase association analysis suggests that a subset of these may be functional during the mitotic phase.
format article
author Elena Panizza
Rui M. M. Branca
Peter Oliviusson
Lukas M. Orre
Janne Lehtiö
author_facet Elena Panizza
Rui M. M. Branca
Peter Oliviusson
Lukas M. Orre
Janne Lehtiö
author_sort Elena Panizza
title Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome
title_short Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome
title_full Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome
title_fullStr Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome
title_full_unstemmed Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome
title_sort isoelectric point-based fractionation by hirief coupled to lc-ms allows for in-depth quantitative analysis of the phosphoproteome
publisher Nature Portfolio
publishDate 2017
url https://doaj.org/article/490425e62f0e446b872c186b12ec9123
work_keys_str_mv AT elenapanizza isoelectricpointbasedfractionationbyhiriefcoupledtolcmsallowsforindepthquantitativeanalysisofthephosphoproteome
AT ruimmbranca isoelectricpointbasedfractionationbyhiriefcoupledtolcmsallowsforindepthquantitativeanalysisofthephosphoproteome
AT peteroliviusson isoelectricpointbasedfractionationbyhiriefcoupledtolcmsallowsforindepthquantitativeanalysisofthephosphoproteome
AT lukasmorre isoelectricpointbasedfractionationbyhiriefcoupledtolcmsallowsforindepthquantitativeanalysisofthephosphoproteome
AT jannelehtio isoelectricpointbasedfractionationbyhiriefcoupledtolcmsallowsforindepthquantitativeanalysisofthephosphoproteome
_version_ 1718388508953935872

Ejemplares similares