Tandem Mass Tag Technology – enabling quantitative protein expression analysis by mass spectrometry (MS)

Thermo Scientific TMT Isobaric Mass Tagging Kits and Reagents enable quantitative labeling of proteins extracted from cells and tissues for identification and analysis by mass spectrometry. Each isobaric tagging 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, 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 powerful tools that enable concurrent identification and quantitation of proteins in different samples using tandem mass spectrometry. Tandem mass tagging (TMT) extends the analytic power of isobaric labeling for MS analysis.

Highlights of Tandem Mass Tag Reagents:

• Powerful – tandem mass tagging enables protein identification and quantitation from multiple samples of cells, tissues or biological fluids
• Robust – consistent chemistry allows efficient transition from method development to multiplex quantitation, enabling biomarker discovery research
• Efficient – amine-reactive NHS-ester activated reagents ensure efficient labeling of membrane and post-translationally modified proteins
• Flexible – expandable system allows concurrent multiplexing of up to six different samples in a single experiment
• 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 for TMT Isobaric Mass Tagging:

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

TMT Product Details:

Isobaric mass tags

Isobaric chemical tags are powerful tools that enable concurrent identification and quantitation of proteins in different samples using tandem mass spectrometry. 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 (Figure).

Our tandem mass tags (TMT) are uniquely designed to enable a rapid and cost-effective transition from method development to high-throughput protein quantitation. The tags consist of TMT⁰ (zero), the TMT² two-plex set and the TMT⁶ six-plex set (Figure). 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.

The TMT method

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.

Procedure summary for MS experiments with Thermo Scientific TMT Isobaric Mass Tagging Reagents. As many as six different samples can be mass-labeled and analyzed in a single mass spectrometry experiment. See Figure for a more detailed description of this procedure.

Analysis summary for TMT experiments for mass spectrometry. 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.

Tandem mass tag MS experimental data

The TMT tags are provided as standalone sets or in optimized kit formats containing all necessary reagents and controls for maximum flexibility, convenience and reliability. The TMT Reagents combined with Thermo Scientific instruments and software provide a complete and integrated solution to perform absolute quantitation of target proteins. Tandem Mass Tag Labeling Technology combined with the industry-leading Thermo Scientific LTQ Orbitrap, LTQ XL and TSQ Quantum Mass Spectrometry Instrument platforms and software provide integrated total system solutions for quantitative protein expression analysis.

• Example data: MS analysis of BSA and other proteins using tandem mass tag technology
• Research poster: Selective enrichment and quantitation of phosphoproteins involved in cell proliferation Major. M., et al., 2008. Thermo Fisher Scientific, Rockford, IL 61101 and Cambridge, MA 02139.

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.

Related Products:

SILAC Protein Quantitation Kits
Glycoprotein Isolation Kits
Phosphopeptide Isolation Kit
Ubiquitin Enrichment Kit
Halt Protease Inhibitor Cocktail
Halt Phosphatase Inhibitor Cocktail

Related Links

2D/Mass Spectrometry Handbook
Tandem Mass Tag Technology Flier
Tandem Mass Tags - opens Proteome Sciences web site

TMT Reagents are protected by U.S. Patent #7,294,456 B2, European Patent EP1275004 B1 and patents pending.

*Trademark; see link in page footer.