image   Compound Identification

All of the IROA peaks are mathematically calculable. Using the IROA Matrix (paired 1:1 mixture of labeled U-5% and 95% 13C fully labeled Internal Standards) the number of carbon in a biological molecule can be determined by the distance between the two monoisotopic peaks, 12C and 13C (see figure below). The relative height of the M+1 and M-1 provides confirmation of this fact, resulting in a triply redundant quality control check point.

The height of the peaks of an isotopically defined compound may be calculated by the binomial expansion of the expression:

When the IROA approach is combined with accurate mass spectrometry measurement for isotopic abundances across a wide linear dynamic range, this facilitates not only the identification of all “non-carbon” atoms in unknown compounds but also reliable quantitative information (the IROA ratio).



Accurate compound identification using Matrix:

1)  Analyze IROA MSMLS authentic compounds standards to get physical data for 600+ compounds. This data is stored in ClusterFinder's internal databases.

2) Run the Matrix sample at least 5 separate injections, ideally from multiple days, to build a dataset.  

3) Perform a search for IROA peaks using ClusterFinder and apply the MSMLS library to the search results.  Hits here become the preferred name for "identified" peaks.  

4) The well-formed IROA Matrix peaks (triply redundant and mathematically calculable) will have the correct formulae assigned.  The next step is to examine all "identified" peaks for adducts and fragments among the unidentified peaks (these are clearly identified as they will have the same IROA shape as parent peaks).  This step picks up a large percentage of the “unidentified” peaks and defines these "identified as related".   These are entered into the database to ensure they will be identified in future analyses (Note: the database automatically annotates the source and all modifications to all records).

5) For peaks not either identified by comparison to a standard or determined to be derived from a standard, the correct formula is assured, and the program will list all isobaric compound names. For this purpose, copies of Pubchem, ChEBI, Lipidmaps, and a peptide database are embedded in the ClusterFinder database (with links to the original source).

6) At any point, when the identity of an unidentified peak is determined, add it into the ClusterFinder database so it will be correctly named in the future (the database will carry the date/time annotation for the new addition).

 A tutorial on IROA Compound Identification can be viewed here.