Measure protein expression in six conditions at simultaneously.
Amine-reactive Thermo Scientific TMT* Isobaric Mass Tagging Kits and Reagents enable multiplex quantitation of proteins extracted from cells and tissues using tandem mass spectrometry.
Tandem Mass Tag* 6-plex (TMTsixplex*) Reagents are sets of isobaric compounds (i.e., same mass and structure, also called isotopomers) that are NHS-activated for covalent, irreversible labeling of primary amines (–NH2) groups. Each isobaric reagent contains a different number of heavy isotopes in the mass reporter region, which results in a unique reporter mass during tandem MS/MS for sample identification and relative quantitation. The reagents label all peptides prepared from cell or tissue samples for analysis of up to six samples in a single MS analysis.
Highlights:
Powerful – concurrent MS analysis of multiple samples increases sample throughput and enables relative quantitation of up to six different samples derived from cells, tissues or biological fluids
Consistent – identical reagent structure and performance among TMTzero*, TMTduplex*, TMTsixplex* and TMT10plex* reagents allows efficient transition from method development to multiplex quantitation, enabling biomarker discovery research across platforms and datasets
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 all peptides regardless of protein sequence or proteolytic enzyme specificity
Compatible – optimized for use with high resolution Thermo Scientific MS/MS platforms, such as LTQ Orbitrap* XL, Orbitrap* Velos Pro*, Orbitrap* Elite, Q Exactive* instruments with data analysis fully supported with Proteome Discoverer* 1.0 and above
Applications:
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
Measurement of 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 quantitation of hundreds to thousands of proteins in a single experiment
Product Details:
Changes in protein expression and post-translational modifications are essential mechanisms of biological regulation and disease. Advancements in liquid chromatography (LC), mass spectrometry (MS) instrumentation and bioinformatics now enable researchers to identify thousands of proteins in a given sample with a high degree of confidence. However, reproducible detection and quantitation of different proteins within samples using mass spectrometry is challenging due to variability in peptide separation, ionization and detection.
Tandem Mass Tag (TMT) Reagents are isobaric chemical tags that enable concurrent identification and quantitation of proteins in different samples using tandem mass spectrometry. The amine-reactive, NHS-ester-activated compounds covalently attach to the peptide amino terminus and free amino termini of lysine residues of peptides and proteins with high efficiency, thereby labeling all peptides in a given sample regardless of enzyme used for digestion. Since TMT reagents share an identical structure and mass (i.e., isotopomers), labeled peptides co-elute during LC separation and are co-isolated during MS/MS analysis, resulting in fewer missing peptide identifications among samples. During MS/MS analysis, each isobaric tag is also fragmented to produce a unique reporter ion mass that is used for sample identification and quantitation. Protein quantitation is accomplished by comparing the relative intensities of the six reporter ions in the MS/MS spectra.
Structural design of the amine-reactive Tandem Mass Tag* Reagents.A. Functional regions of the TMT reagent structure including MS/MS fragmentation sites by higher energy collision dissociation (HCD) and electron transfer dissociation (ETD).B. TMTduplex reagent structures with 13C and 15N heavy isotope positions (red asterisks). C. TMTsixplex reagent structures with 13C and 15N heavy isotope positions (red asterisks).
The Tandem Mass Tag (TMT) Reagent family consists of TMTzero, TMTduplex, TMTsixplex and TMT10plex sets which are specially designed to enable a rapid and cost-effective transition from method development to high-throughput protein quantitation. The TMTzero 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 TMTsixplex reagent set allows sixplex 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.
Procedure summary for MS experiments with Thermo Scientific TMT Isobaric Mass Tagging Reagents. Protein extracts isolated from cells or tissues are reduced, alkylated and digested overnight. Samples are labeled with the TMT Reagents and then mixed before sample fractionation and clean up. Labeled samples are analyzed by LC-MS/MS before data analysis to identify peptides and quantify reporter ion relative abundance.
Analysis summary for TMT Reagent 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.
TMT Reagents are provided as standalone sets or in optimized kit formats containing all necessary reagents and controls for maximum flexibility, convenience and reliability. Tandem Mass Tag Reagents combined with the industry-leading Thermo Scientific instruments and software provide integrated total system solutions for quantitative protein expression analysis.
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.
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):2921-31.
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.
