Once suppression is corrected, Dual MSTUS for accurate quantitation is employed to provide a very accurate mechanism for the normalization of samples against sample-to-sample variances.

  1. The MS Total Usable Signal (MSTUS) normalization algorithm (Warrick) simply assumes that the overall chemical composition of all samples is close enough that the sum of all the verifiable compounds for comparable samples will be “reasonably” constant. Thus, according to this theory if the sum of these verifiable compounds differs then it is more likely that this is due to differences in the physical size (density or similar property) of the original samples. The algorithm was devised to normalize urine samples which often demonstrate very different concentrations or dilutions but has been shown to be equally effective for most classes of solid or liquid samples. In MSTUS, for every sample a “Normalization Factor” (NF) is developed that when the AUCs for all peaks as individually multiplied they will sum to a “common value.”  The “common value” is determined arbitrarily by the experimentalist in every experiment. In the IROA Workflow, we have considered and implemented several key features that have strengthened the MSTUS normalization procedure with the modification we call “Dual MSTUS”:
  2. All verified peaks are, by definition, peaks that are found in both the IS and the sample, i.e., show up in both the C12 envelope and the C13 envelope of a compound’s isotopolog ladder in both the LTRS and experimental samples. This is a very rigorous test of biological relevance.
  3. All the C13 peaks for each compound represent the same amount of material in each sample because they are derived by addition of equal aliquots of the internal standard.
  4. The amplitudes of both the C12 and C13 envelopes suppressed equally are corrected for suppression losses by correcting each according to the same correction factor.
  5. Each sample is represented by two equally important MSTUS values, the C12 MSTUS (the sum of all C12 envelopes), and the C13 MSTUS value (the sum of all C13 envelopes).
  6. The NF is developed to make the C12 MSTUS equal to the C13 MSTUS (which we know to be constant).

Since the C13 MSTUS is always present at the same concentrations this normalization not only avoids any arbitrary quality in the NF but also means that every suppression corrected and normalized sample can be directly compared to any other similarly treated sample, i.e., has the same amount of IS in it.