Understanding the IROA TruQuant protocol

The IROA Peaks and Envelopes

IROA creates distinct signatures in the molecules of a biological samples for identification and quantitation. Molecules are uniformly labeled using both 12C and 13C at approximately 5% for one isotope and approximately 95% for the second isotope. Why 5% or 95%? This is because with traditional (>98%) labeling the M+1/M-1, M+2/M-2 etc. peaks are easily lost, but when enhanced, they can be used for identification and quantitation. the figure below shows the IROA peaks for the 6-carbon molecule arginine.

Green: Arginine C12 envelope peaks labeled with U-5% 13C. Blue: Arginine C13 envelope peaks labeled with U-95% 13C.

Now, let's look at the isotopomeric set or ladder for all isomers of Glutamic acid (C11), below, as seen in RP pos LC-MS. Each isotopolog represents a specific mass by a molecular formula. In the case of carbon isotopologs, these are seen as a collected ladder of peaks extending from the C12 monoisotopic peak to the C13 monoisotopic peaks that differ in mass by 1.00335 amu. All molecules with six carbons will share this ladder but because each formula has a different mass each will begin and end at different masses. Therefore, each isotopolog ladder is unique to and representative of a single formula. (This is true for masses below 800, at a minimum.)

The isotopolog ladders for any formula are unique to that formula, but are common to all its isomers, e.g., Stereo, D/L, Structural, etc. However, the shape of the isotopolog peaks is defined by the relative percentages of the isotopes. The Isotopomeric ladder for Glutamic acid is shared by all molecules that have the same formula (C5H9NO4), including Glutamic acid, O-Actetyl serine, Threo-3-methyl aspartate, and N-Carboxymethyl alanine.

The Figure on the left shows the isotopolog ladder for tryptophan, which contains an equal concentration of “molecules” (1:1) for the right and left side set of peaks; natural abundance on the left, 95% 13C on the right. The relative heights of each isotopolog peak in a ladder is determined by the isotopic balances of the source materials. The height of the peaks of isotopically defined compounds (enriched in a single element such as carbon) may be effectively calculated by the binomial expansion[1] of the expression (12C% + 13C%)N where N equals the number of carbons, and 12C% and 13C% equals the relative isotopic abundance.

In this image we see a situation common in IROA, namely a ladder that has the isotopic signatures contributed from two sources; the first source on the left is the natural abundance compound, and the second source labeled at 95% 13C on the right is its internal standard. These are presented at exactly equal concentrations of molecules from both sources however they are distributed very differently.

[1] This is technically a polynomial expansion in which the dominance of carbon makes the remaining terms less important. See “Addressing the current bottlenecks of metabolomics: Isotopic Ratio Outlier Analysis, an Isotopic labeling technique for accurate biochemical profiling” page 7.

Note that the height of the base peak is never indicative of concentration; rather the sum of all peaks from each collection must be considered. In the case of tryptophan, the base peak is still the C13 monoisotopic peak and represents only about half molecules in the internal standard. The base peak for an IROA compound with more than 20 carbons will no longer be the C13 monoisotopic peak. Instead, depending on the number of carbons, it will become one of the lower mass isotopologs. This is because as the number of atoms in a molecule increases, the probability that the entire molecule contains at least one heavy isotope also increases.

Isotopic Envelopes

All molecules with the same number of carbons will show the same pattern of peaks but will differ in the mass of their monoisotopic peaks according to the remainder of the formula. We refer to these peak height patterns as the peak “isotopic envelopes”. These envelopes are diagnostic for each formula.

The IROA carbon envelope shapes are readily and exactly calculable. The defining feature of the IROA carbon envelope is the mass of both monoisotopic peaks and the mass difference between them. The mass difference between the monoisotopic peaks is always a multiple of the mass of a neutron (~1.00335 amu). The additional peaks discussed above contribute to the extended isotopic envelope (M+2, M+3 etc., M-2, M-3 etc., see Figure 4) and the IROA ClusterFinder software can easily identify these peaks by their mass difference (the mass difference between a 13C and 12C isotope).

Fundamental to the IROA concepts (and inherent in the name Isotopic Ratio Outlier Analysis) is the fact that the ratio of the C-12 envelope to the C-13 envelope is unaffected by suppression even though both the C-12 and C-13 isotopomeric sets may be strongly suppressed. This has afforded a mechanism for suppression correction that has been built into ClusterFinder. Once suppression is corrected, a Dual MSTUS[1] algorithm is employed to provide a very accurate mechanism for the normalization of samples against sample-to-sample variances. This version of ClusterFinder outputs three values: 1) the raw (suppressed) values observed; 2) a suppression-corrected value; and 3) a normalized (suppression-corrected and normalized) value.

[1] Warrack BM, Hnatyshyn S, Ott KH, Reily MD, Sanders M, et al. (2009) “Normalization strategies for metabonomic analysis of urine samples.”, J Chromatogr B Analyt Technol Biomed Life Sci 877: 547–552.

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