Relative quantitation by Tandem Mass Tag (TMT)

Stable Isobaric labelling technologies, such as TMT enables accurate relative quantification of protein abundance of up to 16 samples simultaneously by mass spectrometry-based proteomics.

 

TMT reagents are a series of small chemically reactive stable isotope labelled molecules (usually amine-reactive) which are added (“tagged”) onto peptides following trypsin digestion of the protein samples. Each tagged sample is pooled together as a single sample before being data collection on the mass spectrometer. When quantified, the tags make it possible to determine how much each sample contributed to any particular protein’s abundance, and therefore the abundance protein across the samples.

TMT reagents have three components.

  • A peptide reactive group which binds to the free amines on the peptides via NHS-ester interaction. A cystine reactive TMT reagent is also available.
  • A balance group which ensures all TMTs have the same mass while intact.
  • A reporter group which can be composed of up to 16 different combinations of 13C and 15N isotopes,

Following digestion of the protein samples, individual peptide samples are incubated with the TMT reagents, a different tag used for each sample. After which point the individually tagged samples are then pooled into a single sample. When the sample is sprayed into the MS instrument, the peptides undergo a survey scan (MS1 scan) which in most cases will identify the most abundant peptides within a given timeframe, this appears as the tallest peaks on a chromatogram.

Following the survey scan, the mass spectrometer will then go through each of the peaks one by one, separate the peptide and break it down further in the identify scan (MS2 scan). The fragments produced can be interpreted to figure out the primary structure of the peptide. After this, the MS instrument can break the TMT tags on the peptide in the quantification can (MS3 scan), this causes cleavage of the TMT at the balance group, releasing the reporter group which is then measured by the mass spectrometer.

The unique combination of isotopes allows each tag to appear as a distinct peak making it possible to trace back how much each sample contributed to the total protein content detected in the pool. This allows for a quantifiable comparison of protein content across all samples from a single MS run.

A common challenge with bottom-up MS experiments is dealing with technical variation that occurs between MS runs. This is known as missing values, in which the same sample run twice on the same machine can produce different identities and abundance values because different peptides are missed during the initial survey scan. Missing values make it difficult to quantify differences between samples run separately. As TMT treated samples are scanned simultaneously, the analysis gets around this issue, allowing users to confidently associate the variations seen as differences between the samples rather than fluctuations to normalise against.

Another advantage of TMT is that the tags amplify the signal of low abundance peptides. Pooling the samples increases the amount of peptide being analysed, meaning that low abundance proteins that would be below the level of detection if analysed in isolation, will be boosted to detectable levels.

In addition, by pooling samples together, the number of MS experiments required for up to 16 samples can be reduced to a single run, which saves both cost and time.

The benefits of utilising TMT labels are therefor to obtain data that is easier to quantify, enhance detection of low abundance proteins, and saving money and time by conducting a run.

Pros

  • Allows for more accurate quantitation between multiple samples
  • Pooling samples means less machine time required
  • Higher chance of detecting low abundance proteins

Cons

  • Requires extra preparation steps to accommodate the TMTs
  • MS3 step means less time spent on MS1 and MS2 scans, reducing number of protein IDs found.

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