TMT Quantitation

Introduction

Tandem Mass Tag (TMT) is a powerful technique for comparing protein (and peptide) amounts between six or ten conditions. This technique involves labeling samples after digestion, meaning this technique can be used on in vivo samples, unlike SILAC.

TMT samples are to be submitted as complex mixture samples. It is the responsibility of the customer to ensure that similar amounts of protein is present in each sample and that the samples are contaminant free. A full description of TMT is outside the scope of this website, but details can be found at:

https://en.wikipedia.org/wiki/Tandem_mass_tag
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-mass-spectrometry-analysis/protein-quantitation-mass-spectrometry/tandem-mass-tag-systems.html

Samples to be analyzed by TMT quantitation should be submitted as in-gel complex mixture samples. Analysis of acquired mass-spectrometry data will be performed with Proteome Discoverer 2.1 software.

What Does it Cost?

$1500 for TMT 6-plex, $2000 for TMT 10-plex.

What We Do

We run your TMT samples on our Orbitrap Fusion Lumos mass-spectrometry platforms, using an appropriate LC-MS/MS method. We identify and quantify peptides and proteins from samples using Proteome Discoverer 2.1, and send you a spreadsheet including a list of proteins present and the relative amount of each protein in each sample.

What You Must Do

Prior to submitting TMT samples you should:

  • Contact us before beginning the experiment to discuss experimental design, .
  • Ensure all samples have similar amounts of protein.
  • Run your protein mixtures into the top of the resolving portion of an SDS-PAGE gel. Run in 5-10mm to submit as a single sample.
  • Cut out each stained area as a 5-10mm slice. Each slice will be submitted in a separate tube. Do not cut out the very top of the gel (loading/stacking) area, which is likely to have detergent from your buffer on its surface.
  • Adhere to our guidance when cutting out the band, ensuring you dice each band into 1mm cubes (no smaller) etc. and ensure your slice(s) are no larger than 10mm of gel.
  • Place each slice into an Eppendorf 1.5ml tube, which has been rinsed with 50% organic solvent and millipure water.
  • Follow the general guidelines for sample submission.

Experimental Design

Appropriate experimental design and statistical analysis of results are key to a successful quantitative proteomics experiment. You must contact us before beginning your quantitative experiment. Biological and technical repeats are necessary for robust results, so quantitative experiments can be lengthy and expensive. Careful consideration upfront prevents wasted time and money later.

Any Questions?

If you are planning TMT samples please always contact ProteomicsCore@UTSouthwestern.edu before you prepare and submit your samples.

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