Tag Archives: iTRAQ

Isobaric ions

Two (or more) ions are isobaric if they have the same nominal mass but different exact mass. For example positive radical ions of N2, CO, and C2H4 all have nominal mass of 28 units (z = 1). However, their exact (monoisotopic) masses are 28.00559, 27.99436, and 28.03075 units respectively.

TMT reagents are called isobaric tags, which can be confusing. These labeling reagents within each set (e.g. 2-plex or 6-plex) have the same nominal mass and the same exact mass, therefore they are not isobars as defined by IUPAC.

Ammonium bicarbonate or triethylammonium bicarbonate?

This was a question from one of my blog’s secret readers. Actually, most of the time I feel like I am talking to myself: “Hey Tania, how do you prepare a protein sample for proteolysis?” “Well, Tania, let me show you in a step-by-step tutorial.” No comments, no questions, no pointing out typos, no “thank you, Tania, but there’s a better way to do this”?

Oh well, back to ammonium bicarbonate. This is a volatile salt which breaks down to ammonia, carbon dioxide, and water. Volatile salts are the only salts compatible with MS. Aqueous solutions of ammonium bicarbonate (0.01 – 0.1 M) have pH around 8, the optimal pH for trypsin activity. Ammonium bicarbonate competes with basic amino acids for Coomassie dye, which makes it a great de-staining reagent for the in-gel digestion procedure. All this goodness comes at a very reasonable price – what not to like? Another ammonium salt, triethylammonium bicarbonate (TEAB), is more volatile than ammonium bicarbonate; it is also more expensive. TEAB is a buffer of choice for LC-MS applications: TMT (iTRAQ) amine-reactive labeling, ion-exchange chromatography, protein solubilization (when neutral and acidic pH is undesirable), in-gel digestion, etc.


What is TMT?

TMT (tandem mass tags) are labeling reagents for comparative mass-spectrometry-based proteomics, a Thermo equivalent of iTRAQ. Most popular TMT are amine reactive, but SH-reactive tags are also available. Each isobaric TMT reagent has an amine-reactive NHS-ester group, a spacer arm, and a tandem MS reporter. Either intact proteins or their tryptic digests can be labeled, and up to 6 experimental conditions can be compared in terms of protein expression differences. You can find more information about TMT reagents here.

Our facility is capable of performing both TMT and iTRAQ experiments.