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Relative Quan TMT

Discovery-based relative quantification is an analytical approach that allows the scientist to determine relative protein abundance changes across a set of samples simultaneously and without the requirement for prior knowledge of the proteins involved.

To understand the functions of individual proteins and their place in complex biological systems, it is often necessary to measure changes in protein abundance relative to changes in the state of the system. These measurements have traditionally been performed using Western blot analyses. More recently, modern proteomics has evolved to include a variety of technologies for the routine quantitative analyses of both known and unknown targets. Discovery-based relative quantification is an analytical approach that allows the scientist to determine relative protein abundance changes across a set of samples simultaneously and without the requirement for prior knowledge of the proteins involved. Here we describe three commonly used techniques for relative quantitation of unknown protein/peptide targets using mass spectrometry.

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Overview for TMT-based Quantitation

Isobaric chemical tags are a more universal alternative to SILAC. In a single analysis, they can be used to identify and quantify relative changes in complex protein samples across multiply experimental conditions. They can be used with a wide variety of samples including cells, tissues, and biological fluids. Isobaric chemical tags facilitate the simultaneous analysis of a large number of samples.

Thermo Scientific Tandem Mass Tag (TMT) reagents are isobaric chemical tags consisting of an MS/MS reporter group, a spacer arm and a reactive group. Amine-reactive groups covalently bind to peptide N-termini or to lysine residues. Each tag fragments during MS/MS, producing unique reporter ions. Protein quantitation is accomplished by comparing the intensities of the reporter ions.
 





Literature Highlights



Original TMT paper:

Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS

Thompson A, Schäfer J, et al.
Anal Chem. 2003 Apr 15;75(8):1895-904.


Increasing the Multiplexing Capacity of TMTs Using Reporter Ion Isotopologues with Isobaric Masses

Graeme C. McAlister, Edward L. Huttlin, Wilhelm Haas, Lily Ting, Mark P. Jedrychowski, John C.Rogers, Karsten Kuhn, Ian Pike, Robert A. Grothe, Justin D. Blethrow and Steven P. Gygi
Anal. Chem., 2012, 84 (17), pp 7469–7478



MultiNotch MS3 Enables Accurate, Sensitive, and Multiplexed Detection of Differential Expression across Cancer Cell Line Proteomes

Graeme C. McAlister, David P. Nusinow, Mark P. Jedrychowski, Martin Wühr, Edward L. Huttlin, Brian K. Erickson, Ramin Rad, Wilhelm Haas and Steven P. Gygi
Anal. Chem., 2014, 86 (14), pp 7150–7158


Tracking cancer drugs in living cells by thermal profiling of the proteome

Mikhail M. Savitski, Friedrich B. M. Reinhard, Holger Franken, Thilo Werner, Maria Fälth Savitski, Dirk Eberhard, Daniel Martinez Molina, Rozbeh Jafari, Rebecca Bakszt Dovega, Susan Klaeger, Bernhard Kuster, Pär Nordlund, Marcus Bantscheff and Gerard Drewes
Science 2014, 346(6205)


A comprehensive Xist interactome reveals cohesin repulsion and an RNA-directed chromosome
conformation (Proteomics)


Anand Minajigi, John E. Froberg, Chunyao Wei, Hongjae Sunwoo, Barry Kesner, David Colognori, Derek Lessing, Bernhard Payer, Myriam Boukhali, Wilhelm Haas and Jeannie T. Lee
Science 2015, 349(6245)