TMTpro-134C & TMTpro-135N Label Reagents, 1 x 5 mg - FAQs

View additional product information for TMTpro 18-plex, TMTpro-134C, and TMTpro-135N Label Reagents - FAQs (A58335, A52047, A52046, A52048, A52045)

18 product FAQs found

Can the TMT and TMTpro reagents be mixed?

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

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Can TMTpro label reagents dissolved in anhydrous acetonitrile be dried down and stored for later use?

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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Can I combine TMTpro tags with TMT tags?

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

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If I already have TMTpro 16-plex tags, what do I need to order to get a total of 18-plex TMTpro tags?

Depending upon the number of vials and amount of each tag in the vial for the TMTpro 16-plex tags you already have, you will have to purchase the appropriate TMTpro-134C and TMTpro-135N Label Reagents.
- If you already have TMTpro 16plex Label Reagent Set, 1 x 5 mg (Cat. No. A44520), you will have to purchase TMTpro-134C and TMTpro-135N Label Reagents, 1 x 5 mg (Cat. No. A52046).
- If you already have TMTpro 16plex Label Reagent Set, 6 x 0.5 mg (Cat. No. A44522), you will have to purchase TMTpro-134C and TMTpro-135N Label Reagents, 6 x 0.5 mg (Cat. No. A52048).

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What is provided with the TMTpro-134C & TMTpro-135N Label Reagents?

TMTpro-134C & TMTpro-135N Label Reagents provide only two TMTpro tags, TMTpro-134C and TMTpro-135N. Cat. No. A52046 provides TMTpro-134C and TMTpro-135N tags as 1 vial each of 5 mg and Cat. No. A52048 provides TMTpro-134C and TMTpro-135N tags as 6 vials each of 0.5 mg.

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Can I purchase the TMTpro-134C and TMTpro-135N Label Reagents separately from the TMTpro 18-plex Label Reagent Set?

Yes, Cat. No. A52046 provides TMTpro-134C and TMTpro-135N tags as 1 vial each of 5 mg and Cat. No. A52048 provides TMTpro-134C and TMTpro-135N tags as 6 vials each of 0.5 mg.

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What are the components of the TMTpro 18-plex Label Reagent Sets?

The TMTpro 18-plex Label Reagent Sets are the combination of corresponding TMTpro 16 plex Label Reagent Sets and TMTpro-134C & TMTpro-135N Label Reagents. Cat. No. A52045 is a combination of Cat. No. A52046 and Cat. No. A44520 where all TMTpro tags are provided as 1 vial each of 5 mg. Cat. No. A52047 is a combination of Cat. No. A52048 and Cat. No. A44522 where all TMTpro tags are provided as 6 vials each of 0.5 mg.

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At which step should I label my mass spec samples with TMT reagents?

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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Do I have to apply correction factors for analyzing data for TMT/TMTpro-labeled samples?

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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How do I bridge the data from different sample sets for TMT-based quantitation?

This is most often done with the utilization of a "pool channel" for each multiplex set. This reference channel typically contains an equimolar mixture of all samples which is labeled with one of the TMT tags that is shared among the sets. The pool channel can be used to then normalize relative protein abundance measurements across multiplexed sets.

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In the EasyPep sample preparation workflow, it is mentioned that TMT/TMTpro reagent can be added to peptides before or after the clean-up step. Are there any differences between these two methods, regarding the labeling efficiency or relative quantitation?

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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What type of mass spectrometer can I use for Tandem Mass Tag (TMT) analysis?

We recommend using high-resolution Orbitrap Tribrid (e.g., Fusion, Lumos, Eclipse), Orbitrap Exploris (e.g., 240, 480), or Q Exactive (e.g,. Plus, HF, HFX) series of instruments.

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What is the main purpose of Tandem Mass Tag (TMT) reagents?

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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Is TMT quantitation relative or absolute?

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

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What is the main purpose of TMT (Tandem Mass Tag) reagents?

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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When should I use quadrupole versus ion trap isolation?

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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How effective is the salt removal using Pierce Peptide Desalting Spin Columns?

With Pierce Peptide Desalting Spin Columns, more than 90% of salt or labeling tags such as TMT tags can be removed.

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What is the reproducibility of Pierce Peptide Desalting Spin Columns from sample to sample?

With Pierce Peptide Desalting Spin Columns, the reproducibility coefficient of variation (CV) is ± 20%.

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