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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

Workflow 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 multiple 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.



Workflows_TNT_1115.jpg


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)


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.
 


Workflows_TMT_102616.jpg




Literature Highlights



Original TMT paper:

Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtur...

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 acr...

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
conformatio...


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)

Sample Preparation

Sample Prep Workflow for TMT-based Quantitation

 

A typical TMT-based quantification experiment involves proteolytic digestion, usually with trypsin, labeling each sample with Tandem Mass Tag (TMT) reagents and then combining the samples prior to LC-MS/MS analysis. Each TMT reagent is composed of a reactive NHS-ester group, a spacer arm and an MS/MS reporter. They are small molecules with identical structure that covalently attach to the free amino termini of lysine residues of peptides and proteins, thereby labeling various peptides in a given sample. TMT labeling increases the number of samples analyzed, and peptides identified and quantified, in a single analysis. These reagents label peptides prepared from a variety of sources, including cell-based or tissue samples, ranging from two to six individual samples for simultaneous quantitative comparison.

The sample preparation for isolating proteins can vary widely depending on the goal of the experiments.  The mixing of the samples following digestion and labeling permits a variety of fractionation techniques including phosphopeptide enrichment or strong cation exchange fractionation. These techniques can improve the detection of abundance changes for both low-abundance peptides and post-translational modifications such as phosphorylation and/or glycosylation.

A step-by-step TMT labeling procedure for intact proteins as well as peptides including preparing whole cell protein extracts, protein digestion, and peptide labeling can be found here.



Related Products


Link to download Protein Quantitation Using Mass Spectrometry handbook.

A wide selection of TMT kits are commercially available. The kits contain all of the reagents needed for comparing two samples in small profiling studes to six samples in complex analyses with multiple conditions (e.g. time courses, dose responses, replicates, and multiple-sample comparisons). TMT kits can be combined with many Thermo Scientific peptide enrichment technologies.

A detailed description for all TMT products can be found here.

Mass Spectrometry

Mass Spectrometry Workflow for TMT-based Quantitation



The key benefit of TMT-based quantitation is that multiple samples, of any origin, can be quantified in a single analysis. As peptides from multiple samples labeled with TMT reagents are chemically identical and isobaric, they co-elute during online separation and are detected simultaneously during MS analysis as a single indistinguishable precursor ion peak. Following fragmentation, the tags from each individual sample generate unique low-mass reporter ions. Protein quantitation is then accomplished by comparing the intensities of the reporter ions and peptide identification is accomplished using the peptide fragment ions in the same MS/MS spectrum.

Methods for traditional TMT experiments involve isolating precursor ions and generating peptide fragment ions and TMT reporter ions in a single spectrum using higher-energy collisional dissociation. HCD performed on Orbitrap mass spectrometers has been shown to produce superior quality MS/MS data for both identification and quantitation.1 This triple quadrupole-like fragmentation produces more high-intensity, accurate-mass, low-m/z reporter ions than other technologies, resulting in enhancement of both accuracy and precision of quantitation. The accuracy of the results is very dependent on the quality of the pre-fractionation and LC separation, which is used to reduce the number of co-eluting species which will be co-isolated and fragmented together. This is particularly important for samples of high complexity or high dynamic range. As quantitation is performed at the MS/MS level, quantitation interference from co-isolated species will be indistinguishable.2 High MS-level resolving power can be used to resolve the co-eluting isobaric species to help detect contamination. High MS/MS-level resolving power can be used to resolve reporter ions from interfering ions for accurate quantitation. The superior isolation ability of ion traps allows isolation window of 0.7 Da or quadrupoles on Q Exactive platforms with isolation windows of 0.7 Da improving quantitation accuracy for samples without loss of sensitivity. However, in most cases MS2 based quantitation is better suited for samples of low complexity.

The development of a novel method called synchronous precursor selection (SPS) overcomes the issue with contamination at the same time enables quantitation of complex samples. This approach is exclusive to Orbitrap Fusion and Orbitrap Fusion Lumos Tribrid mass spectrometers. In this approach, the parent ion is selected in MS1 scan, isolated in the quadrople and fragment by CID in the ion trap. Upon fragmentation, multiple MS2 fragment ions are selected and isolated using single trap fill and waveform (synchronous precursor selection). Up to 20 fragments can be isolated simultaneously. Selected MS2 fragment ions are transferred back into the IRM and HCD fragmentation is performed. MS3 fragments are detected in the Orbitrap for the most accurate MS3 based TMT quantitation. Using SPS dramatically increases the signal intensity and improves the ratio accuracy (due to counting statistics) and at the same time dramatically boosts sensitivity increasing the total number of peptides quantified significantly. Using this approach, multiplexed quantification of peptides in a variety of biological samples is possible and this workflow is only possible on the Orbitrap Fusion and Orbitrap Fusion Lumos mass spectrometers.

For TMT-based peptide quantitation, columns at least 25 cm in length with LC gradients equal or greater than two hours and optimal sample loads are recommended in order to achieve base-peak separation, reproducibility and minimize overlapping precursor ions.


 

References

 

1. High precision quantitative proteomics using iTRAQ on an LTQ Orbitrap: a new mass spectrometric m...

Köcher T, Pichler P, et al.
J Proteome Res. 2009 Oct;8(10):4743-52.

 

2. MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics

Ting L, Rad R, et al.
Nat Methods. 2011 Oct 2;8(11):937-40.
 


 

Additional Resources


Webinar: How to do TMT experiments on Orbitrap based mass spectrometers. From sample preparation to ...

Xiaoyue Jiang
Thermo Fisher Scientific
Webinar

 

How to do TMT experiments on Orbitrap based mass spectrometers. From sample preparation to data acqu...
 

Xiaoyue Jiang and Rosa Viner
Thermo Fisher Scientific
Slide Presentation



RELATED PRODUCTS

 

Orbitrap Elite

Orbitrap Fusion


Orbitrap Fusion Lumos


Q Exactive Plus


Q Exactive HF


 

Thermo Scientific provides UPLC/HPLC systems that perform at low nano, micro, and high flow rate regimes to meet a wide variety of experimental needs.  Thermo Scientific EASY-nLC 1200 and Dionex UltiMate® 3000 RSLCnano LC systems use split-free designs to achieve exceptional stability and reproducibility and they easily couple to all Thermo Scientific mass spectrometers.

Data Analysis

Data Analysis Workflow for TMT-based Quantitation


Thermo Scientific Proteome Discoverer software version 2.1 is recommended for quantitative analysis of TMT-based data. Quantitation is performed at the MS/MS level for traditional HCD experiments, or at the MS3 level for SPS MS3 experiments, by comparing the relative S/N abundances of the TMT reporter ions. Proteins are identified using the accurate-mass precursor information combined with the fragmentation data. The relative peak abundances of multiple distinct peptides from each protein are then averaged to determine the change in protein abundance in the treated samples.


For detailed step-by-step information about quantitative analysis using Proteome Discoverer™ software and to download a free 60-day demonstration version of Proteome Discoverer software version 2.1, please visit the Thermo Scientific Proteomics Software Portal.

Grant Central

Grant Central Resources for TMT-based Quantitation


Every research idea matters. At Thermo Fisher Scientific, we are dedicated to helping you advance your research, and that includes becoming your scientific partner in supporting your grant application efforts.  Our latest grant writing resources are listed below.

Need supporting information for your grant proposal or have a grant writing related question? Visit Grant Central or Contact Us.
 


GENERAL Resources

 

Top 5 reasons to upgrade from a Thermo Scientific™ Hybrid Orbitrap™ to a Thermo Scientific™ Tribrid™...
Grant Application Resource


Achieving robust, accurate TMT quantitation efficiency with Tribrid technology

Grant Application Resource

Technical Resources

 

High Throughput Quantitative Proteomics Using Isobaric Tags: Presentation of real-world applications...
White Paper


How to do TMT experiments on Orbitrap based mass spectrometers. From sample preparation to data acqu...
Slide Presentation


TMT SPS MS3 in publications: Synchronous Precursor Selection (SPS)

MS3 Publication List
 

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