iTRAQ® Reagents: Can Samples Labeled with Both the 4-Plex and 8-Plex Kits Be Mixed?

For research use only. Not for use in diagnostic procedures.

Answer

SCIEX does not recommend mixing samples labeled with the reagents from the two separate iTRAQ® kits, 4plex and 8plex, due to the differences in the reagent chemistry.

This picture below shows the 4plex iTRAQ Reagent with the reporter group (114-117 amu) and the balancing group 31 - 28 amu) that together form an isobaric tag (145 amu) and ensure that each peptide has the same mass when analyzed by TOF MS or Q1 mass mode. Peptides labeled with the 4plex reagent will have a signal that is 4-times more intense. In MS/MS mode, peptides labeled with this reagent undergo fragmentation, and then the peptide reporter tag ions and the total peptide mass can be measured for relative quantitative analysis.

The 8plex iTRAQ Reagent kit contains eight different reporter tag labels and a different balancing group ranging from 184-192 amu. Therefore, the 8plex reporter groups are not an extension of the 4plex reporter groups; the 8plex kit uses an entirely different combination of labels for the reporter and balance groups. Mixing the samples labeled with the 4plex and 8plex reagents, therefore, would not work in TOF MS,  because you would have different masses for the same peptide sequence.

Instead of mixing the 8plex and the 4plex reagents together, try the following: Mix a 114 reporter tag from a 4plex kit with another 114 tag from a 4plex kit if you take into consideration the volumes needed to make 1 unit. This creates a new problem of lot traceability.