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Issue 17, 2015
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Multiplexing strategy for simultaneous detection of redox-, phospho- and total proteome – understanding TOR regulating pathways in Chlamydomonas reinhardtii

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Abstract

New methods for studying the complexity of multiple PTMs in functional proteomics are required to understand cell signaling processes. In this study, a multiplexing 2DE-based approach is introduced for parallel analysis of the redox-, phospho-, and total-proteome. This triplexing approach uses spectrally distinct fluorophores, is not matrix-specific and requires relatively low sample amounts with applicability to any cell/tissue type. This methodology was applied for the study of Target of Rapamycin (TOR) regulating pathways in Chlamydomonas reinhardtii. With emerging research demonstrating a complex yet unclear relationship between TOR kinase, autophagy, and lipid metabolism, rapamycin treatment was used to induce TOR inhibition in C. reinhardtii and redox-, phospho- and total proteome changes were assessed using the triplexing approach. We identified a total of 68 spot abundance changes in response to TOR inhibition which provide a basis for understanding this highly conserved, master regulator in algae.

Graphical abstract: Multiplexing strategy for simultaneous detection of redox-, phospho- and total proteome – understanding TOR regulating pathways in Chlamydomonas reinhardtii

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Supplementary files

Article information


Submitted
27 Feb 2015
Accepted
29 May 2015
First published
29 May 2015

This article is Open Access

Anal. Methods, 2015,7, 7336-7344
Article type
Paper

Multiplexing strategy for simultaneous detection of redox-, phospho- and total proteome – understanding TOR regulating pathways in Chlamydomonas reinhardtii

S. P. Rodrigues, S. Alvarez, E. G. Werth, W. O. Slade, B. Gau, E. B. Cahoon and L. M. Hicks, Anal. Methods, 2015, 7, 7336
DOI: 10.1039/C5AY00521C

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