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On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics
Date Issued
2014-04-16
Date Available
2016-04-08T11:17:23Z
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.
Sponsorship
European Commission - Seventh Framework Programme (FP7)
Science Foundation Ireland
Type of Material
Journal Article
Publisher
MDPI
Journal
Biology
Volume
3
Issue
2
Start Page
320
End Page
332
Language
English
Status of Item
Peer reviewed
This item is made available under a Creative Commons License
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On-Beads Digestion in Conjunction with Data-Dependent Mass.pdf
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518.66 KB
Format
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104
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