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
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. 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
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
Tandem Mass Tags
consist of TMT0 (zero), the TMT2 two-plex set and
the TMT6 six-plex set
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
TMT0 has no isotopic substitutions and is
used for method development.
A pair of isobaric mass labels with a single
isotopic substitution per tag is used for simple
pairwise comparisons of relative protein expression.
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
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. 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.
summary for TMT Reagent experiments for mass
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.
Figure5. Protein Profiling with TMT Tags
tandem mass tagging enables protein
identification and quantitation from multiple
samples of cells, tissues or biological fluids
consistent chemistry allows efficient
transition from method development to multiplex
quantitation, enabling biomarker discovery
amine-reactive NHS-ester activated reagents ensure efficient labeling of membrane and post-translationally modified proteins
expandable system allows concurrent
multiplexing of up to six different samples in a
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
Protein identification and quantitation from
multiple samples of cells, tissue or biological
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
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.
et al. (2008). Relative
quantification of proteins in human
cerebrospinal fluids by MS/MS using 6-plex
isobaric tags. Anal. Chem. 80(8), 2921
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.