TMT® Quantitation

Tandem Mass Tagging (TMT) is one of two main methods of labeled quantitation in proteomics (the other being iTRAQ). Like iTRAQ, TMT uses tags of the same mass. During the TMT labeling reaction, the amine group of the tag labels the lysine residue or N-terminus of peptides. After MS/MS fragmentation, the resulting reporter ions have different isotopic masses, thus allowing relative quantitation. Please see the table below to compare TMT and iTRAQ.

Applied Biomics has optimized our TMT protocol to significantly increase the sensitivity of detecting low abundant proteins and the total number of proteins:

  • Optimized sample preparation on samples such as serum, clinical samples with interfering substances, protein complex and whole cell lysate
  • Optimized a fractionation protocol specifically targeting the low-abundant basic proteins (pI > 8.0), such as membrane proteins, or a certain protein mass/pI range of your choice
  • Extended nanoLC gradient coupled with optimized MSMS parameters, totaling a 12 hour instrument time

We have optimized protein extraction for TMT analysis on the following sample types:

  • Whole cell lysate from cultured cells, bacteria and other organisms
  • Subcellular organelles such as Mitochondria, Lysosome and Exosome etc
  • Protein complex such as IP or column purified complex with very low protein amount (<2ug)
  • Clinical samples with interfering substances such as high salt in urine
  • Serum samples with high abundant proteins and a wide dynamic range

TMT experimental procedure

iTRAQ procedure: step 1 - protein extraction and buffer exchange

Protein samples

iTRAQ procedure: step 2 - Reduction/Alkylation & Trypsin Digestion

Reduction/Alkylation & Trypsin Digestion

TMT reagent labeling kit

TMT reagent labeling

TMT mix containing 126 and 131 reporter ions

Mix and clean up samples

iTRAQ procedure: step 5 - peptide separation by Nano-HPLC


iTRAQ procedure: step 6 - peptide finger printing by MS


iTRAQ procedure: step 7 - peptide fragmentation by MSMS


Ratio of 126 to 131 reporter ions

Reporter ion

TMT vs iTRAQ table

TMT and iTRAQ are both effective methods of labeled proteomics quantitation. While TMT offers more reagents and number of comparisons, iTRAQ provides absolute quantitation in addition to relative quantitation. The following table comparese these two platforms:

Labeling typeIsobaricIsobaric
QuantitationRelative onlyAbsolute & Relative
Total mass of tag229 (TMT 6-plex)145 (iTRAQ 4-plex)
Reporter masses126-131 (TMT 6-plex)114-117 (iTRAQ 4-plex)
Available reagents2-plex, 6-plex,
10-plex, 11-plex
4-plex, 8-plex

Sample Info

Sample type: We accept in-solution, in-gel samples or IP beads for this service.

Protein amount: 5-200 µg of total protein from each sample.

Protein concentration: 5-20 mg/ml is preferred for in-solution samples..

Buffer: Please feel free to submit samples in whichever buffer that you would normally use.  Please inform our scientists about buffer composition. Additional charge may apply on samples that require extensive amount of the extra work such as protein eluting, concentrating, buffer exchange or serum abundant protein depletion.

Biohazardous Materials: We do NOT accept samples containing any biohazardous materials. If your samples contain any biohazardous materials, please make sure to deactivate them completely using the appropriate procedure and declare them in the order form. In addition, we do NOT accept any samples containing Level 4 biohazardous materials even if they are deactivated. Please view here for the rank of biohazardous materials.

Sample type: Please inform our scientists about your sample type. We will send you additional tips for each sample type.


Services DescriptionAcademic/Governmental LabsIndustrial/Commercial Labs
TMT® Experiment350Acall for price350Ncall for price

Turnaround time is 7-9 days. Price covers the following:

  • Reduction and alkylation
  • Trypsin digestion
  • TMT labeling followed by sample cleaning up
  • Nano-LC coupled with auto-spotting
  • MS and MS/MS
  • Standard database search against one species in NCBI or SwissProt database using SequestHT
  • Data report (see sample report below)

Price does NOT cover sample preparation or the cost of TMT kit.