Cargando…

On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics

With the advent of the “-omics” era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a si...

Descripción completa

Detalles Bibliográficos
Autores principales: Turriziani, Benedetta, Garcia-Munoz, Amaya, Pilkington, Ruth, Raso, Cinzia, Kolch, Walter, von Kriegsheim, Alexander
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4085610/
https://www.ncbi.nlm.nih.gov/pubmed/24833512
http://dx.doi.org/10.3390/biology3020320
_version_ 1782324688524935168
author Turriziani, Benedetta
Garcia-Munoz, Amaya
Pilkington, Ruth
Raso, Cinzia
Kolch, Walter
von Kriegsheim, Alexander
author_facet Turriziani, Benedetta
Garcia-Munoz, Amaya
Pilkington, Ruth
Raso, Cinzia
Kolch, Walter
von Kriegsheim, Alexander
author_sort Turriziani, Benedetta
collection PubMed
description With the advent of the “-omics” era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a single protein. It is therefore crucial to investigate the regulation of proteins in the context of a pathway first and signaling network second. In this study, we demonstrate that a quantitative interaction proteomic approach, combining immunoprecipitation, in-solution digestion and label-free quantification mass spectrometry, provides data of high accuracy and depth. This protocol is applicable, both to tagged, exogenous and untagged, endogenous proteins. Furthermore, it is fast, reliable and, due to a label-free quantitation approach, allows the comparison of multiple conditions. We further show that we are able to generate data in a medium throughput fashion and that we can quantify dynamic interaction changes in signaling pathways in response to mitogenic stimuli, making our approach a suitable method to generate data for system biology approaches.
format Online
Article
Text
id pubmed-4085610
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-40856102014-07-08 On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics Turriziani, Benedetta Garcia-Munoz, Amaya Pilkington, Ruth Raso, Cinzia Kolch, Walter von Kriegsheim, Alexander Biology (Basel) Article With the advent of the “-omics” era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a single protein. It is therefore crucial to investigate the regulation of proteins in the context of a pathway first and signaling network second. In this study, we demonstrate that a quantitative interaction proteomic approach, combining immunoprecipitation, in-solution digestion and label-free quantification mass spectrometry, provides data of high accuracy and depth. This protocol is applicable, both to tagged, exogenous and untagged, endogenous proteins. Furthermore, it is fast, reliable and, due to a label-free quantitation approach, allows the comparison of multiple conditions. We further show that we are able to generate data in a medium throughput fashion and that we can quantify dynamic interaction changes in signaling pathways in response to mitogenic stimuli, making our approach a suitable method to generate data for system biology approaches. MDPI 2014-04-16 /pmc/articles/PMC4085610/ /pubmed/24833512 http://dx.doi.org/10.3390/biology3020320 Text en © 2014 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 license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Turriziani, Benedetta
Garcia-Munoz, Amaya
Pilkington, Ruth
Raso, Cinzia
Kolch, Walter
von Kriegsheim, Alexander
On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics
title On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics
title_full On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics
title_fullStr On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics
title_full_unstemmed On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics
title_short On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics
title_sort on-beads digestion in conjunction with data-dependent mass spectrometry: a shortcut to quantitative and dynamic interaction proteomics
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4085610/
https://www.ncbi.nlm.nih.gov/pubmed/24833512
http://dx.doi.org/10.3390/biology3020320
work_keys_str_mv AT turrizianibenedetta onbeadsdigestioninconjunctionwithdatadependentmassspectrometryashortcuttoquantitativeanddynamicinteractionproteomics
AT garciamunozamaya onbeadsdigestioninconjunctionwithdatadependentmassspectrometryashortcuttoquantitativeanddynamicinteractionproteomics
AT pilkingtonruth onbeadsdigestioninconjunctionwithdatadependentmassspectrometryashortcuttoquantitativeanddynamicinteractionproteomics
AT rasocinzia onbeadsdigestioninconjunctionwithdatadependentmassspectrometryashortcuttoquantitativeanddynamicinteractionproteomics
AT kolchwalter onbeadsdigestioninconjunctionwithdatadependentmassspectrometryashortcuttoquantitativeanddynamicinteractionproteomics
AT vonkriegsheimalexander onbeadsdigestioninconjunctionwithdatadependentmassspectrometryashortcuttoquantitativeanddynamicinteractionproteomics