Reducing False Positives from Isotopic Misidentification in AS-MS

In affinity selection-mass spectrometry (AS-MS), one of the more challenging-to-discern causes of false positives is compound misidentification due to isotopic pattern overlap.  These errors occur when the X+1 of one compound is perceived as the C12 of another compound.  This can also happen at the X+2 level.  This error is particularly perilous because it can affect both the original hit as well as the background hit.

Experimental

Scientists used Analytical Studio’s automated X–1 and X–2 cross-hit filter check during isotopic pattern matching to determine its impact on reducing false positives.  

Key Finding

Enabling the automated X–1 and X–2 cross-hit filter check during isotopic pattern matching resulted in a 5% reduction in confirmed hits.  This implies that 5% of the initial reported hits were likely false positives caused by isotopic confusion.

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Recommended Practice

Always enable X–1 and X–2 misidentification checks during compound assignment.  If your AS-MS data processing software doesn’t have this capability, perhaps it’s time to look for alternative tools.  This simple processing setting can eliminate a significant portion of false positives at the source, improving the quality of your final hit list.