TMTpro™ 135CD Label Reagent, 1 x 5 mg - FAQs

The TMTpro 32plex and TMTpro Deuterated Label Reagents are guaranteed for one year from the date of receipt when stored at the recommended temperature in original, unopened packaging.

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You would need to use TMTpro 16plex Label Reagent Set (Cat. No. A44520) in combination with TMTpro 16plex Deuterated Label Reagent Set (Cat. No. A40000817).

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Yes, 134C and 135CD Label Reagents (Cat. No. A40000853) provides TMTpro-134C and TMTpro-135CD tags in two separate vials with each vial containing 5 mg of the tag in lyophilized format. 135CD Label Reagent (Cat. No. A40000818) provides one vial containing 5 mg of TMTpro-135 CD tag in lyophilized format.

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134C and 135CD Label Reagents (Cat. No. A40000853) provides two TMTpro tags, TMTpro-134C and TMTpro-135CD. The tags are supplied in two separate vials, with each vial containing 5 mg of the tag in lyophilized format.

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TMTpro 135CD is the only tag offered as a standalone product (Cat. No. A40000818, 135CD Label Reagent).

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The TMTpro 32plex and TMTpro Deuterated Label Reagents can be purchased in four different size formats:
- Cat. No. A40000817: 16plex Deuterated Label Reagent Set, 1 x 5 mg (per tag)
- Cat. No. A40000839: 32plex Label Reagent Matched Set, 1 x 5 mg (per tag); Note that this is a combo kit of TMTpro 16plex Label Reagent Set (Cat. No. A44520) and TMTpro 16plex Deuterated Label Reagent Set (Cat. No. A40000817)
- Cat. No. A40000853: 134C and 135CD Label Reagents, 1 x 5 mg (per tag)
- Cat. No. A40000818: 135CD Label Reagent, 1 x 5 mg (per tag)

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No, the TMT and TMTpro reagents cannot be mixed because the reporter ions are in the same region.

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Yes, unused TMTpro label reagent that has been dissolved in anhydrous acetonitrile can be aliquoted into tubes, dried under vacuum at 25-30 degrees C, purged with nitrogen, and stored in a sealed bag with desiccant at -20 degrees C.

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We do not recommend mixing TMTpro and TMT tags, as they differ in chemical structure.

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TMT labeling can be performed after reduction, alkylation, digestion, or prior to cleanup, if the sample is buffered at appropriate pH (8-8.5) in a suitable buffer without primary amines (e.g., Tris, glycine). TMT labeling can also be performed after peptide clean-up. Labeling of peptides after clean-up enables measuring and normalizing of the peptide samples for equal mixing.

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Applying correction factors is required for more accurate relative protein quantitation as the isotopic impurity is variable among the tags, ranging from 5-10%.

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The EasyPep kit chemistry is fully compatible with TMT/TMTpro reagents. Most commonly, TMT/TMTpro reagents are used to label peptides immediately after digestion and before peptide cleanup. This enables combining samples for a single cleanup step which reduces variability for more reproducible quantitative measurements. Another option is to label samples after peptide cleanup. This approach provides efficient labeling of the samples but may require the use of a peptide quantification assay to ensure that samples are equally mixed before LC-MS analysis. Both the workflows should provide high labelling efficiencies using our recommended TMT/TMTpro reagent to sample ratios.

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Labeling efficiency can be determined for individual labeled samples by searching for peptides using TMT/TMTpro as a variable (i.e., dynamic) modification peptide amino terminus and lysines. The ratio of peptide to tag by mass (w:w) should be 1:4 to 1:8 for complete labeling of most samples.

Low labeling efficiency can also be caused by improper storing or handling of TMT/TMTpro reagents that leads to hydrolysis of -NHS groups on the tag, rendering the reagents to be less reactive.

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TMT reagents and TMTpro reagents are different chemicals with different masses. Therefore, we do not recommend mixing the two tags as the combined samples would be significantly more complex resulting in fewer quantified proteins.

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Label-free, TMT reagents, and TMTpro reagents can be used with any protein sample type. SILAC and Neucode quantitation can only be used with cell lines and model organisms that can be metabolically labeled.

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SILAC can be used to multiplex 2 to 3 samples. TMT reagents can be used to multiplex 2, 6, 10, and 11 samples. TMTpro reagents can be used to measure up to 16-18 samples simultaneously. 32 samples can be measured using a combination of TMTpro non-deuterated and deuterated tags.

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Tandem Mass Tag technology is used to individually label different protein samples so they can be combined into a single sample for LC-MS analysis. The major advantages of this workflow are higher sample throughput, less instrument analysis time, high precision of peptide quantitation among replicates, and fewer missing quantified proteins among different samples.

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TMT quantitation is relative between samples unless a heavy peptide standard is included for comparison.

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TMT technology is used to perform multiplex quantitation of proteins extracted from cells and tissues. Rather than quantify single proteins (for example with an antibody), here, large numbers of proteins can be quantified in a single run.

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Quadrupole isolation is faster because ions must travel through the quadrupole analyzer regardless of the analysis type chosen. As a result, there is little to no time penalty when using the quadrupole for MS² isolation. In such a case, quadrupole transmission efficiency will depend on isolation width, m/z, and the cleanliness of the analyzer. Ion trap isolation is best suited for MS³ experiments, including those performed using multinotch isolation for TMT analysis; these types of isolation events are not possible with the quadrupole analyzer.

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