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Biology 2014, 3(2), 320-332; doi:10.3390/biology3020320
Article

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

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Received: 9 January 2014 / Revised: 13 March 2014 / Accepted: 25 March 2014 / Published: 16 April 2014
(This article belongs to the Special Issue Advances in Proteomics Methods)
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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.
Keywords: interaction proteomics; in solution digest; label-free quantification; proteomic; mass spectrometry; protein identification; systems biology interaction proteomics; in solution digest; label-free quantification; proteomic; mass spectrometry; protein identification; systems biology
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Turriziani, B.; Garcia-Munoz, A.; Pilkington, R.; Raso, C.; Kolch, W.; von Kriegsheim, A. On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics. Biology 2014, 3, 320-332.

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