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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.
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.
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. TMT kits can be combined with many Thermo Scientific peptide enrichment technologies.
A detailed description for all TMT products can be found here. 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. 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.
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. The superior isolation ability of ion traps allows isolation window of 0. 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. 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. 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.
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