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

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Title: On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics
Authors: Turriziani, Benedetta
Garcia Munoz, Amaya
Pilkington, Ruth
Raso, Cinzia
Kolch, Walter
Kriegsheim, Alexander von
Permanent link: http://hdl.handle.net/10197/7554
Date: 16-Apr-2014
Abstract: 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.
Funding Details: European Commission - Seventh Framework Programme (FP7)
Science Foundation Ireland
Type of material: Journal Article
Publisher: MDPI
Keywords: Interaction proteomics;In solution digest;Label-free quantification;Proteomic;Mass spectrometry;Protein identification;Systems biology
DOI: 10.3390/biology3020320
Language: en
Status of Item: Peer reviewed
Appears in Collections:Conway Institute Research Collection
SBI Research Collection
Medicine Research Collection

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