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.
Find additional tips, troubleshooting help, and resources within our Mass Spectrometry Support Center.
