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  Quantitative Proteomics with Tandem Mass Tags (TMT®) Reagent

Introduction:

Amine-reactive Thermo Scientific TMT* Isobaric Mass Tagging Kits and Reagents enable quantitative tandem labeling of proteins extracted from cells and tissues for identification and analysis by mass spectrometry.

Each isobaric Tandem Mass Tag* Reagent is composed of an amine-reactive NHS-ester group, a spacer arm and an MS/MS reporter. The reagents label peptides prepared from cell-based or tissue samples, and make a structure M-F-N-(R)-peptide, either two samples for the duplex kit or six samples for the sixplex kit. For each sample, a unique reporter mass results in the MS/MS spectrum, providing a simple means of identification.

Changes in protein expression and post-translational modifications are essential mechanisms of biological regulation and disease. Advancements in mass spectrometry (MS) instrumentation, bioinformatics and quantification methods, such as label-free quantification, metabolic labeling and chemical tagging, now enable researchers to identify and quantitatively analyze thousands of proteins in a given sample with a high degree of accuracy.

Isobaric chemical tags are tools that enable concurrent identification and quantitation of proteins in different samples using tandem mass spectrometry. This procedure is somewhat like the iTRAQ quantitative proteomics analysis. They are small chemical 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 (Figure1).

Figure1. Structure of a Tandem Mass Tag

Each member has a unique mass and reports sample-specific abundance of a labeled peptide during MS/MS analysis.

Cleavable linker: Preferentially fragments under typical MS/MS conditions to release the mass reporter.

Mass normalizer: Each member has a unique mass that balances the mass reporter, ensuring the same overall mass for all tags in a set.

Reactive group: Reactive NHS ester provides high-efficiency aminespecific labeling of proteins/peptides.

Tandem Mass Tags consist of TMT0 (zero), the TMT2 two-plex set and the TMT6 six-plex set (Figure2). The TMT0 tag allows testing and optimization of sample preparation, labeling, fractionation and MS fragmentation for peptide identification and reporter detection without using the more costly isotope-labeled compounds. The TMT2 reagent set allows two-plex protein profiling for small studies. The TMT6 reagent set allows six-plex protein profiling for multiple conditions, including time courses, dose responses, replicates or multiple sample comparisons. Each TMT tag is based on the same chemical structure, eliminating the need to modify labeling conditions or HPLC separation conditions between experiments.

Figure2. The TMT family of isobaric tag reagents

A. TMT⁰ has no isotopic substitutions and is used for method development.

B. A pair of isobaric mass labels with a single isotopic substitution per tag is used for simple pairwise comparisons of relative protein expression.

C. A six-plex of isobaric mass labels each with five isotopic substitutions per tag is used. Used for complex analyses including multi-plex patient screening, time-course analysis or dose escalation studies.

During the MS/MS analysis, each isobaric tag produces a unique reporter ion signature that makes quantitation possible. In the first MS analysis, the labeled peptides are indistinguishable from each other; however, in the tandem MS mode during which peptides are isolated and fragmented, each tag generates a unique reporter ion. Protein quantitation is then accomplished by comparing the intensities of the six reporter ions in the MS/MS spectra. (Figure3-5)

Figure3. Procedure summary for MS experiments with TMT Isobaric Mass Tagging Reagents

As many as six different samples can be mass-labeled and analyzed in a single mass spectrometry experiment.

The relative abundance of the target protein or peptide fragment in six different samples is easily measured by comparing the signature mass peaks generated by the different mass tags.

Highlights:

1. Powerful – tandem mass tagging enables protein identification and quantitation from multiple samples of cells, tissues or biological fluids
2. Robust – consistent chemistry allows efficient transition from method development to multiplex quantitation, enabling biomarker discovery research
3. Efficient – amine-reactive NHS-ester activated reagents ensure efficient labeling of membrane and post-translationally modified proteins
4. Flexible – expandable system allows concurrent multiplexing of up to six different samples in a single experiment
5. Compatible – optimized fragmentation and fully supported quantitation with Proteome Discoverer* 1.0 for all Thermo Scientific LC MS/MS platforms, such as LTQ XL* and LTQ Orbitrap* XL Systems

Applications:

1. Protein identification and quantitation from multiple samples of cells, tissue or biological fluids
2. Protein expression profiling of normal vs. disease states or control vs. treated
3. Multiplex up to six different samples concurrently in a single experiment
4. Quantitative analysis of proteins for which no antibodies are available
5. Identification and quantitation of membrane and post-translationally modified proteins
6. Identification and quantification of hundreds to thousands of proteins in a single experiment

References:

1. Thompson, A., et al. (2003). Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem.75 (8), 1895-1904.

2. Dayon, L., et al. (2008). Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. Anal. Chem. 80(8), 2921 -2931.
3. Nilsson, C.L. et al. (2010). Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1alpha signaling network: an initial study in GSC11 glioblastoma stem cells. J. Proteome Res. 9:430-43.