TMTpro™ 10plex Isobaric Label Reagent Set (Unit Mass Reporter), 1 x 5 mg - FAQs

查看更多产品信息 TMTpro™ 10plex Isobaric Label Reagent Set (Unit Mass Reporter), 1 x 5 mg - FAQs (A40000928)

15 个常见问题解答

What are the primary applications of the TMTpro 10plex Isobaric Label Reagent Set (Unit Mass Reporter), 1 x 5 mg (Cat. No. A40000928)?

The TMTpro 10plex Isobaric Label Reagent Set (Unit Mass Reporter), 1 x 5 mg (Cat. No. A40000928) enables high-precision quantitative proteomics across a broad range of MS platforms. It features reagents with a 1 Da mass difference between reporter ions, making it ideal for use on non-Orbitrap instruments with low MS/MS resolving power, such as the Thermo Scientific Stellar series MS instruments and various MS instruments from other manufacturers.

This reagent set also supports 10plex quantification using the complementary ion cluster in MS2 spectra, which can enhance quantification accuracy and precision on Orbitrap and non-Orbitrap platforms. For more detailed information, please refer to the following publications:
https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00813
https://pubmed.ncbi.nlm.nih.gov/39725028/

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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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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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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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I have low levels (<95%) of TMT-labeling for my samples. What should I check?

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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Can I use TMT reagents in combination with TMTpro reagents?

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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What sample types can I use with different mass spec quantitation systems?

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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How many samples can I combine for simultaneous, multiplex proteomic mass spec quanitification?

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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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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