Using Reporter Ion Isotopic Distribution Values to Correct for Impurities - Proteome Discoverer Software - Using Reporter Ion Isotopic Distribution Values to Correct for Impurities - Proteome Discoverer Software - Proteome Discoverer Software

iTRAQ and TMT kits consist of labels that contain different numbers of ¹³C atoms, ¹⁵N atoms, or both. For simplicity, assume that a 4plex kit yields peaks at 114, 115, 116, and 117 m/z, which correspond to ¹³C1, ¹³C2, ¹³C3, and ¹³C4. Because the label substances are not 100 percent isotopically pure, each label contains a certain number of other atoms. For example, the 116 label would not consist only of label molecules having three ¹³C atoms but might also contain label molecules with only one or two ¹³C atoms or even four or five ¹³C atoms. As a result, these impurities lead to an observed peak at 116 m/z, which is smaller than might be expected if the tag were 100 percent isotopically pure, and additional peaks at positions –2, –1, +1, +2 Da apart from 116 m/z. The intensities of the latter peaks are proportional to the amount of the described isotopic impurities. When the 116 label and the 114, 115, and 117 labels are used, these latter three labels contribute to the peak at 116 m/z because of their isotopic impurities.

The intensity of the peak at 116 m/z effectively includes the following contributions:

(observed intensity 116) = (true intensity 116) – (intensity loss because of 116 impurities) + (intensity gain because of other label impurities)

To obtain the true intensity value of the 116 label—that is, the amount of the substance initially labeled with the 116 tag—you must correct the experimentally observed peak for the impurity of the labels.

For a 4plex sample, four formulas use the above equation for each of the labels, and the proper correction would consider both contributions in the formula by solving the system of coupled linear equations:

(intensity_of_loss_because_of_116_impurity and intensity_of_gain_because_of_other_label_impurities)

For this correction, you must enter the isotopic distribution of each of the labels used in the quantification method, as described in Excluding PSMs with high levels of coisolation. The values are part of each of the iTRAQ or TMT label kits that were used.

You can also deconvolve the overlapping labels using other methods. Compatible with the Mascot search engine, the application uses a first-order approximation to the solution. The error made is small when the intensities of all possible contributing labels are of similar height, and it becomes larger if the intensity differences become larger.

You can find more information on this topic, at www.matrixscience.com/help/quant_config_help.html.

In the Sections menu, choose Component > Correction > Type AB Certificate.

In TMT kits, the values of impurities in reporter ion labels are normalized to the highest peak. In the application, they are normalized to 100 percent, which the application considers to be the sum of all impurities.

For TMT 6plex kits, using correction factors is optional, because the resolution in Q-Exactive or Orbitrap Elite instruments is enough to separate ¹³C and ¹⁵N isotopes.

TMT 10plex kits use no correction factors by default. You can add correction factors to these kits by following the instructions in Excluding PSMs with high levels of coisolation.

In the kits, correction factors are the same for the same batch of isotopes and are usually valid for three to five years. See Checking the quantification method.