Managing Isomers in Affinity Selection-Mass Spectrometry: The Hidden Challenge in Library Design

Isomers are a frequent and complex challenge in affinity selection-mass spectrometry (AS-MS).  Because isomers share identical molecular masses, they are indistinguishable by m/z alone yet may behave differently in solution. Depending on library design and data processing strategy, isomers can introduce false positives, redundant hits, or ambiguous assignments.

Experimental

Scientists looked at a typical AS-MS compound library of 250,000 compounds to determine the number of unique molecular formulae.  The fewer unique molecular formulae present in the library, the greater the odds that there would be isomer-related miss-identifications.

Key Finding

In the experimental library, only 66,000 unique molecular formulae were represented, meaning each formula appeared on average 3–4 times across the library.  As this library was plated across only 96 wells, the duplication of molecular formulae means that there would be isomer-related complications.  While some of these complications can be handled by library design and plating, as the compression ratio in AS-MS experiments increases, isotopic interference is more likely to be prevalent.

Analytical Studio’s unique cross-hit filter analysis now includes enhanced capabilities to filter out isomers.  By treating each isotope peak individually, isotopic contribution to false positives can be significantly reduced or eliminated.

Recommended Practice

Isomers affect both the design and interpretation of AS-MS screens.  Poor handling leads to inflated hit counts, loss of chemical diversity, or missed opportunities.  Carefully planning your library, using intelligent processing settings, and automated cross-hit analysis capabilities that consider each isotopic peak individually is critical to successfully analyze these libraries.