TMTpro™ 18-plex Label Reagent Set, 1 x 5 mg - FAQs

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

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

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